Synthetic peptide and its uses

ABSTRACT

The invention provides a fragment of C1q which is characterized in that a plurality of such fragments selectively binds immune complexes or aggregated immunoglobulins in the presence of monomeric immunoglobulin. The invention also provides a synthetic peptide comprising the sequence: 
     
         Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr
 
    
      1               5                   10
                  SEQ ID NO 2!
 
     or variants thereof capable of binding immunoglobulin. Like the C1q fragment, a plurality of the peptides can selectively bind immune complexes or aggregated immunoglobulins in the presence of monomeric immunoglobulin. As a result of this property, the fragments and peptides are well-adapted for removing immune complexes and aggregated immunoglobulins from fluids containing monomeric immunoglobulin, and for detecting or quantitating immune complexes in such fluids. The invention also provides a binding material for removing immune complexes or aggregated immunoglobulins from a fluid. The binding material comprises plural binding peptides, the peptides being characterized in that a plurality of them selectively binds immune complexes and aggregated immunoglobulins in the presence of monomeric immunoglobulin. The invention also provides methods of treating an inflammatory response and prolonging the survival of transplanted tissue.

This application is a continuation-in-part of application Ser. No.08/335,049, filed Nov. 7, 1994, now U.S. Pat. No. 5,698,449 which was adivision of application Ser. No. 07/598,416, filed Oct. 16, 1990, nowU.S. Pat. No. 5,364,930.

FIELD OF THE INVENTION

This invention relates to a fragment of C1q and to a synthetic peptideand to their use to selectively bind immune complexes and aggregatedimmunoglobulins. The invention also relates to a binding material whichcan be used to selectively remove immune complexes and aggregatedimmunoglobulins from fluids. The invention further relates to methods oftreating inflammation and prolonging the survival of transplantedtissue.

BACKGROUND OF THE INVENTION

An immune complex is an aggregate of immunoglobulins, non-immunoglobulinserum proteins, and antigens. Immune complexes are formed as a naturalconsequence of the immune response to antigens of infectious agents, tonormal tissue components in the case of autoimmune diseases, totumor-associated antigens, and to other antigens. The complexes arenormally removed by the cells of the reticulo-endothelial system. Whenthis system is compromised or overloaded, circulating immune complexesmay deposit in a number of organs, thereby causing possibly severeclinical problems. Further, in cancer, it is postulated that immunecomplexes may block other effector mechanisms of the immune system whichwould otherwise destroy malignant cells. Several studies have indicatedthat removal of circulating immune complexes may be an effectivetherapeutic technique. See, e.g., Theofilopoulos and Dixon, Adv.Immunol., 28, 90-220 (1979); Theofilopoulos and Dixon, Immunodiagnosticsof cancer, page 896 (M. Decker Inc., New York, N.Y. 1979).

Immune complexes form as a result of immunoglobulins reacting withantigens. Immunoglobulins are able to cross-link antigens so that alattice network of immunoglobulins bound to antigens is formed. Once theantigen-immunoglobulin reaction has occurred, the immune complex canthen be decorated with a variety of serum proteins such as the proteinsof the complement cascade.

Complement component C1q selectively binds immune complexes in thepresence of monomeric immunoglobulin because of the molecule's abilityto develop a "functional affinity" when binding immune complexes. A"functional affinity" results when multiple low affinity receptors,confined in space, interact with multiple ligands, which are alsoconfined in space. Normally, an individual ligand would rapidlyassociate and dissociate from the low affinity receptor but, whenmultiple ligands in a complex interact with multiple receptors, thedissociation from the receptors is very slow since the probability ofall ligands dissociating at the same time is very low. The slowerdissociation rate results in an affinity several orders of magnitudegreater than the individual receptor's affinity. The difference inaffinities for the individual ligand and the complexed ligand produces aselection for the complexed ligand when presented with both species.

The mature C1q molecule contains two distinct portions, the stalk andthe globular head. There are six globular head regions per C1q molecule.Each contains a low affinity immunoglobulin binding site. Hughes-Jonesand Gardner, Immunology, 34, 459-63 (1978); Duncan and Winter, Nature,332, 738-40 (1988). Since there are six globular head regions on C1q,the molecule can form multiple binding interactions with the multipleimmunoglobulins present in immune complexes. Id. The result is a highernet affinity for immune complexes (id.) due to the low probability ofmore than one bound globular head receptor dissociating simultaneously(i.e., a functional affinity develops). Thus, when C1q is presented withboth immune complexes and monomeric immunoglobulin, it selectively bindsto the immune complexes because of the slower dissociation kinetics ofthe immune complexes.

Many investigators have tried to identify the residues onimmunoglobulins that are recognized by C1q. Initial theoretical studiesthat compared the sequences of immunoglobulin Fc regions of variousspecies known to bind human C1q produced four possible sites in twogeneral locations: 1) the residues flanking Trp277 and Tyr278 (residues275-295) (Lukas et al., J. Immunol., 127, 2555-60 (1981); Prystowsky etal., Biochemistry, 20, 6349-56 (1981)); and 2) the residues flankingGlu318 (residues 316-338) (Stalinheim et al., Immunochem., 10, 501-507(1973); Burton et al., Nature, 288, 338-44 (1980)). Various studies byauthors advocating one or the other site produced conflicting results.

However, Duncan and Winter recently performed a series of moreconclusive experiments. Duncan and Winter, Nature, 332, 738-40 (1988).Using recombinant DNA techniques, they were able to systematically alterthe various residues of the two disputed sites. Then, by determining theability to bind C1q of each of the resulting immunoglobulins, the actualsite and specific binding residues were determined. They localized thecore of the C1q interactions to residues 318, 320, and 322 in the Fcregion of human IgG. Despite, the success of Duncan and Winter, the siteon immunoglobulins where C1q binds may not be limited to the residuesindicated by their work. In fact, other immunoglobulin residues may alsobe involved in the C1q-immunoglobulin interaction that could not bedetected using their approach. This will not be resolved until highresolution x-ray diffraction data are obtained for the C1q-Fc regioncomplex and the complete binding interaction is determined.

Bacterial proteins such as Staphylococcus aureus Protein A also bind tothe immunoglobulin Fc region. Unlike C1q, the Protein A-immunoglobulininteraction is understood in detail. In a series of crystallographicstudies by Deisenhofer, et al., the structure of human IgG Fc, ProteinA, and finally the IgG Fc-Protein A Fragment B co-crystal weredetermined. Deisenhofer et al., Hoppe-Seyler's Z. Physiol. Chem. Bd.,359, S. 975-85 (1978); Marquart et al., J. Mol. Biol., 141, 369-91(1980). One of the most important pieces of information to come fromthis structure is the exact contact residues involved in theinteraction. Those residues are Met 252, Ile 253, Ser 254, Val 308, Leu309, His 310, Gln 311, Asn 312, His 433, Asn 434, His 435, and Tyr 436of the human IgG Fc region. These residues are located at the interfacebetween the CH2 and CH3 regions of the Fc portion of IgG, and some ofthem (309-312) are in close proximity to the proposed immunoglobulinbinding site for C1q (318, 320 and 322).

Unlike C1q, Protein A binds to the Fc portion of immunoglobulins withhigh affinity. Ellman, Arch. Biochem. Biophys., 74, 443-450 (1958).Thus, Protein A cannot differentiate between complexed and monomericimmunoglobulins.

However, PCT application WO 89/04675 teaches the preparation of analogsof Protein A that have a lower affinity for the Fc region and which candevelop a functional affinity for immune complexes when arrayed in aspecific manner. The analogs are analogs of a binding domain of ProteinA or of related sequences from functionally similar bacterial proteinssuch as Protein G (see page 10). This PCT application reports thatoligomers of the analogs, or an array of the analogs disposed about thesurface of an insoluble matrix, develop a functional affinity for immunecomplexes.

SUMMARY OF THE INVENTION

The invention provides a fragment of C1q which is characterized in thata plurality of such fragments selectively binds immune complexes oraggregated immunoglobulins in the presence of monomeric immunoglobulin.The fragment can be used to bind immune complexes or aggregatedimmunoglobulins. It can also be used to detect or quantitate immunecomplexes or to remove immune complexes or aggregated immunoglobulinsfrom fluids. In particular, since a plurality of the fragments canselectively bind immune complexes or aggregated immunoglobulins in thepresence of monomeric immunoglobulin, the fragment is especiallywell-adapted for removing immune complexes and aggregated immunoglobulinfrom fluids containing monomeric immunoglobulin and for detecting orquantitating immune complexes in such fluids.

The invention also provides a synthetic peptide comprising the sequence:

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr     1               5                   10                      SEQ ID NO 2!

or variants thereof capable of binding immunoglobulin. The syntheticpeptide can be used to bind immune complexes or aggregatedimmunoglobulins, to detect or quantitate immune complexes, or to removeimmune complexes or aggregated immunoglobulins from fluids. Like the C1qfragment, a plurality of the peptides can selectively bind immunecomplexes or aggregated immunoglobulins in the presence of monomericimmunoglobulin. As a result of this property, the peptides of theinvention are also well-adapted for removing immune complexes andaggregated immunoglobulin from fluids containing monomericimmunoglobulin, and for detecting or quantitating immune complexes insuch fluids.

The C1q fragment and the synthetic peptide can be prepared in a numberof ways, including using recombinant DNA techniques. Accordingly, theinvention also comprises a DNA molecule encoding the C1q fragment or thesynthetic peptide, a vector comprising the DNA molecule operativelylinked to expression control sequences, and a host cell transformed withthe vector. The C1q fragment or synthetic peptide can be prepared byculturing the transformed host cell.

The invention further provides a test kit for detecting or quantitatingimmune complexes. The kit comprises a container of the C1q fragment or acontainer of the synthetic peptide.

Also, the invention provides a binding material for removing immunecomplexes or aggregated immunoglobulins from a fluid. The materialcomprises plural binding peptides, the peptides being characterized inthat a plurality of them selectively binds immune complexes andaggregated immunoglobulins in the presence of monomeric immunoglobulin.Preferably, the binding peptides are the C1q fragments of the inventionor synthetic peptides comprising the sequence

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr     1               5                   10                      SEQ ID NO 2!

or a combination of the two. Immune complexes and aggregatedimmunoglobulins can be removed from fluids containing them by contactingthe fluids with the binding material at a temperature and for a timesufficient to bind the immune complexes and aggregated immunoglobulinsto the material, and then separating the fluid from the material. Theinvention also provides a device for removing immune complexes oraggregated immunoglobulins from a fluid comprising the binding materialand a means for encasing the material so that the fluid can be contactedwith it.

Further, the synthetic peptide or C1q fragment can be used to treat aninflammatory response in a mammal by administering to the mammal aneffective amount of the C1q fragment or synthetic peptide. "Treat" isused herein to mean that the inflammatory response is reduced orprevented. The synthetic peptide or C1q fragment can also be used toprolong the survival of tissues transplanted into a mammal byadministering to the mammal an effective amount of the C1q fragment orsynthetic peptide. The C1q fragment and synthetic peptide exert theseeffects because they inhibit the binding of C1q to immunoglobulin boundto antigen, thereby preventing the initiation of the complement cascade.It has been found that other peptides inhibit the binding of C1q toimmunoglobulin and can, consequently, be used to treat an inflammatoryresponse and to prolong the survival of transplanted tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shown are the sequences (from top to bottom) of S. aureus ProteinA fragment B, the predicted helical region of C1q, and CBP2.

FIG. 2 Helical wheel diagrams for the helical region of Protein A(residues 142-153), predicted helical region of C1q B chain (residues189-200), and CBP2 Peptide (SEQ ID NO:3).

FIG. 3 Shows the elution profile of CBP2 on a Sephadex G25 (cross-linkeddextran, fractionation range 1000-5000 M_(r)) column.

FIG. 4A Reverse phase HPLC chromatograph of CBP2 under reducingconditions.

FIG. 4B Reverse phase High Performance Liquid Chromatography (HPLC)chromatograph of CBP2 under nonreducing conditions.

FIG. 4C Reverse phase HPLC chromatograph of CBP2 in the presence of anoxidizing agent to favor the formation of disulfide linked CBP2peptides.

FIG. 5 Shows the inhibition of the binding of immunogloblin labeled withhorseradish peroxiclase Ig-HRP) to solid phase Protein A by CBP2,Protein A, or lutenizing hormone releasing hormone (LHRH).

FIG. 6 Shows the inhibition of the binding of rabbit Ig-HRP to solidphase C1q by CBP2, C1q, or LHRH.

FIG. 7 The elution profile of horseradish peroxidase/anti-horseradishperoxidase immune complexes (PAP) passed over the HiPAC™ LTQ-CBP2column.

FIG. 8 The elution profile of a mixture of PAP plus monomeric rabbitIgG.

FIG. 9A Elution of PAP on a CBP2 column washed with solution of CBP2.

FIG. 9B Elution profile of PAP on a CBP2 column washed with a solutionof LHRH.

FIG. 10 The elution profile of PAP on the HiPAC™ LTQ-CBP2 column whenPAP were incubated with either CBP2 or LHRH prior to being loaded on thecolumn.

FIG. 11 The elution profile of aggregated human IgG on the HiPAC™LTQ-CBP2 column.

FIG. 12 The elution profile of aggregated human IgG plus monomeric humanIgG.

FIG. 13A The elution profile of biotinylated aggregated human IgG plusmonomeric human IgG (total IgG).

FIG. 13B The elution profile of biotinylated aggregated human IgG.

FIG. 13C The elution profile of C1q binding material.

FIG. 14A The elution profile of biotinylated aggregated human IgG indiluted pooled normal human plasma (total IgG).

FIG. 14B Elution profile of aggregated IgG.

FIG. 14C Elution profile of C1q binding material.

FIG. 15 Graph of the ratio of C1q binding material to Protein A bindingmaterial for five patient sera prior to passage over the HiPAC™ LTQ-CBP2column.

FIG. 16 Graph of the ratio of C1q binding material to Protein A bindingmaterial for five patient sera after passage over the HiPAC™ LTQ-CBP2column.

FIG. 17 Shows the elution profile of aggregated human IgG diluted in C1qbuffer and passed over the HiPAC™ FPLC-CBP2 column.

FIG. 18 Shows the elution profile of biotinylated aggregated human IgGdiluted in normal human plasma and passed over the HiPAC™ FPLC-CBP2column.

FIG. 19 Graph of the ratio of C1q binding material to Protein A bindingmaterial for five patient sera after passage over the HiPAC™ FPLC-CBP2column.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The C1q fragment of the invention may be any fragment of a C1q molecule,a plurality of which selectively binds immune complexes and aggregatedimmunoglobulins in the presence of monomeric immunoglobulin. Thefragment may be a fragment of a C1q molecule of any species, such ashuman or rabbit. Suitable fragments may be identified as described inExample 1 below. The C1q fragment identified in Example 1 has thesequence

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr,     1               5                   10                      SEQ ID NO 2!

and other suitable fragments may be identified by examining the aminoacid sequences of C1q molecules for sequences homologous to this C1qfragment.

The invention also provides synthetic peptides comprising the sequence:

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr     1               5                   10                      SEQ ID NO 2!

or variants thereof capable of binding immunoglobulin. The syntheticpeptides are also characterized in that a plurality of them selectivelybinds immune complexes and aggregated immunoglobulins in the presence ofmonomeric immunoglobulin.

The most preferred synthetic peptide has the sequence:

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln     1               5                   10    Ala Thr Leu Leu Cys                     15                      SEQ ID NO 3!

The sequence

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr

of SEQ ID NO 3 is that of the C1q fragment identified above (SEQ ID NO2). The two leucine residues were added to the carboxyl terminus of thisfragment as spacing residues to separate the active residues from afuture solid phase support (see below). The cysteine residue was addedto the carboxyl terminus to allow for coupling of the peptide to solidphases. This preferred synthetic peptide is identified herein as CBP2.

As used herein, a "synthetic peptide" means a peptide which is not anaturally-occurring peptide, although "synthetic peptides" may bealtered versions of naturally-occurring peptides. "Synthetic peptides"include peptides synthesized in vitro and peptides synthesized in vivo.In particular, "synthetic peptides" include peptides produced intransformed host cells by recombinant DNA techniques.

As used herein, "variant" means a synthetic peptide having changes(additions, deletions, or substitutions) in the specified amino acidsequence, provided that the "variant" synthetic peptide still has theability to bind immunoglobulin. "Variants" can have a higher or loweraffinity for immunoglobulin than the specified sequence, but allsynthetic peptides according to the invention are characterized in thata plurality of them will selectively bind immune complexes andaggregated immunoglobulins in the presence of monomeric immunoglobulin.Preferred "variants" are those in which changes in the specifiedsequence are made so that the resulting sequence will assume an alphahelical structure when modeled by secondary structure predictionprograms.

The C1q fragment or synthetic peptide may be made in a variety of ways.For instance, solid phase synthesis techniques may be used. Suitabletechniques are well known in the art, and include those described inMerrifield, in Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotiseds. 1973); Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Davis etal., Biochem. Int'l, 10, 394-414 (1985); Stewart and Young, Solid PhasePeptide Synthesis (1969); U.S. Pat. No. 3,941,763; Finn et al., in TheProteins, 3rd ed., vol. 2, pp. 105-253 (1976); and Erickson et al. inThe Proteins, 3rd ed., vol 2, pp. 257-527 (1976). Solid phase synthesisis the preferred technique of making individual C1q fragments andsynthetic peptides since it is the most cost-effective method of makingsmall peptides.

The C1q fragment and synthetic peptide may also be made in transformedhost cells using recombinant DNA techniques. To do so, a recombinant DNAmolecule coding for the fragment or peptide is prepared. Methods ofpreparing such DNA molecules are well known in the art. For instance,sequences coding for the C1q fragment could be excised from C1q genesusing suitable restriction enzymes. Alternatively, the DNA moleculecould be synthesized using chemical synthesis techniques, such as thephosphoramidite method. Also, a combination of these techniques could beused.

The invention also includes a vector capable of expressing the C1qfragment or synthetic peptide in an appropriate host. The vectorcomprises the DNA molecule that codes for the C1q fragment or syntheticpeptide operatively linked to appropriate expression control sequences.Methods of effecting this operative linking, either before or after theDNA molecule is inserted into the vector, are well known. Expressioncontrol sequences include promoters, activators, enhancers, operators,ribosomal binding sites, start signals, stop signals, cap signals,polyadenylation signals, and other signals involved with the control oftranscription or translation.

The vector must contain a promoter and a transcription terminationsignal, both operatively linked to the DNA molecule coding for the C1qfragment or synthetic peptide. The promoter may be any DNA sequence thatshows transcriptional activity in the host cell and may be derived fromgenes encoding homologous or heterologous proteins (preferablyhomologous) and either extracellular or intracellular proteins, such asamylases, glycoamylases, proteases, lipases, cellulases and glycolyticenzymes.

The promoter may be preceded by upstream activator and enhancersequences. An operator sequence may also be included downstream of thepromoter, if desired.

The vector should also have a translation start signal immediatelypreceding the DNA molecule, if the DNA molecule does not itself beginwith such a start signal. There should be no stop signal between thestart signal and the end of the DNA molecule coding for the C1q fragmentor the synthetic peptide.

Expression control sequences suitable for use in the invention are wellknown. They include those of the E. coli lac system, the E. coli trpsystem, the TAC system and the TRC system; the major operator andpromotor regions of bacteriophage lambda; the control region offilamentaceous single-stranded DNA phages; the expression controlsequences of other bacteria; promoters derived from genes coding forSaccharomyces cerevisiae TPI, ADH, PGK and alpha-factor; promotersderived from genes coding for Aspergillus oryzae TAKA amylase and A.niger glycoamylase, neutral alphaamylase and acid stable alpha-amylase;promoters derived from genes coding for Rhizomucor miehei asparticproteinase and lipase; and other sequences known to control theexpression of genes of prokaryotic cells, eukaryotic cells, theirviruses, or combinations thereof.

The vector must also contain one or more replication systems which allowit to replicate in the host cells. In particular, when the host is ayeast, the vector should contain the yeast 2 u replication genes REP1-3and origin of replication.

The vector should further include one or more restriction enzyme sitesfor inserting the DNA molecule coding for the C1q fragment or syntheticpeptide and other DNA sequences into the vector, and a DNA sequencecoding for a selectable or identifiable phenotypic trait which ismanifested when the vector is present in the host cell ("a selectionmarker").

Suitable vectors for use in the invention are well known. They includepUC (such as pUC8 and pUC4K), pBR (such as pBR322 and pBR328), pUR (suchas pUR288), phage λ and YEp (such as YEp24) plasmids and derivativesthereof.

In a preferred embodiment, a DNA sequence encoding a signal orsignal-leader sequence, or a functional fragment thereof, is included inthe vector between the translation start signal and the DNA moleculecoding for the C1q fragment or synthetic peptide. A signal orsignal-leader sequence is a sequence of amino acids at the aminoterminus of a polypeptide or protein which provides for secretion of theprotein or polypeptide from the cell in which it is produced. Many suchsignal and signal-leader sequences are known.

By including a DNA sequence encoding a signal or signal-leader aminoacid sequence in the vectors of the invention, the C1q fragment orsynthetic peptide encoded by the DNA molecule may be secreted from thecell in which it is produced. Preferably, the signal or signal-leaderamino acid sequence is cleaved from the fragment or peptide during itssecretion from the cell. If not, the fragment or peptide shouldpreferably be cleaved from the signal or signal-leader amino acidsequence after its isolation.

Signal or signal-leader sequences suitable for use in the inventioninclude Saccharomyces cerevisiae alpha factor (see U.S. Pat. No.4,546,082), S. cerevisiae a factor (see U.S. Pat. No. 4,588,684), andsignal sequences which are normally part of precursors of proteins orpolypeptides such as the precursor of interferon (see U.S. Pat. No.4,775,622).

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityto it of the C1q fragment or synthetic peptide encoded for by the DNAmolecule, rate of transformation, ease of recovery of the C1q fragmentor synthetic peptide, expression characteristics, bio-safety and costs.A balance of these factors must be struck with the understanding thatnot all hosts may be equally effective for the expression of aparticular DNA sequence.

Within these general guidelines, useful microbial hosts include bacteria(such as E. coli sp.), yeast (such as Saccharomyces sp.) and otherfungi, insects, plants, mammalian (including human) cells in culture, orother hosts known in the art.

Next, the transformed host is cultured under conventional fermentationconditions so that the desired C1q fragment or synthetic peptide isexpressed. Such fermentation conditions are well known in the art.

Finally, the C1q fragment or synthetic peptide is purified from theculture. These purification methods are also well known in the art.

The C1q fragment or synthetic peptide may be utilized to bind aggregatedimmunoglobulin or immune complexes. The C1q fragment or the syntheticpeptide may be added directly to a fluid containing the aggregatedimmunoglobulin or immune complexes, or may be attached to a solid phasebefore being contacted with the fluid containing the aggregatedimmunoglobulin or immune complexes.

As noted above, a plurality of the C1q fragments or of the syntheticpeptides selectively binds to immune complexes or aggregatedimmunoglobulins in the presence of monomeric immunoglobulin. This makesthe C1q fragments and synthetic peptides particularly well-adapted tobind immune complexes and aggregated immunoglobulins in fluidscontaining monomeric immunoglobulin and for detecting or quantitatingimmune complexes in such fluids. Fluids which may contain immunecomplexes or aggregated immunoglobulins, as well as monomericimmunoglobulin, include body fluids (such as blood, plasma and serum)and reagent and pharmaceutical products (such as animal sera, solutionsof gamma globulin, isolated blood components, solutions containingmonoclonal antibodies, etc.).

Individual C1q fragments and synthetic peptides have a low affinity forimmunoglobulins, including those in immune complexes and aggregatedimmunoglobulins. Accordingly, the plurality of C1q fragments orsynthetic peptides must be held in sufficient proximity to each other sothat multiple points of attachment to the immune complex or aggregatedimmunoglobulins can be made and a functional affinity formed (see thediscussion of functional affinity in the Background section). This canbe accomplished by forming oligomers of the C1q fragments or syntheticpeptides (i.e., multiple copies of the C1q fragment or synthetic peptideon the same molecule) or by attaching the C1q fragments or syntheticpeptides to a solid phase at an effective density.

On the oligomers, the C1q fragments and synthetic peptides will bespaced an adequate distance apart to permit the formation of multiplepoints of attachment with immune complexes and aggregatedimmunoglobulins. As a result, the oligomer will have a higher affinityfor immune complexes and aggregated immunoglobulins than for monomericimmunoglobulin (i.e., a functional affinity). If necessary, amino acidspacers can be used between the C1q fragments and synthetic peptides toachieve the proper spacing. The oligomers can be prepared in the sameways as described above for the C1q fragment and synthetic peptide. Theoligomers may also be attached to a solid phase as described below.

C1q fragments or synthetic peptides may also be attached to a solidphase at an effective density. An effective density is one that allowsthe immunoglobulin binding sites of the C1q fragments and syntheticpeptides to be spaced apart on the surface of the solid phase in such amanner as to permit multiple point attachment with the immune complexesor aggregated immunoglobulins. At this density, the C1q fragments andsynthetic peptides will bind immune complexes and aggregatedimmunoglobulins in preference to monomeric immunoglobulin because afunctional affinity develops. A density which is so low that the spacingof the C1q fragments or synthetic peptides exceeds the distance betweenbinding sites on the immune complexes or aggregated immunoglobulins mustbe avoided. The density of C1q fragment or synthetic peptide which worksbest can be determined empirically and will depend on such factors asthe surface area of the solid phase material, mode of coupling, thespecific nature of the C1q fragment or synthetic peptide used, and thesize of the immune complexes or aggregated immunoglobulins.

The C1q fragments, synthetic peptides and oligomers may be attached toany known solid phase material. For the C1q fragments and syntheticpeptides, a solid phase which has a relatively nonporous surface ispreferably used. Since the C1q fragments and synthetic peptides aresmall molecules, it is believed that they may become attached to thesolid phase in the pores of a porous material. They may, therefore, bindimmune complexes or aggregated immunoglobulins less readily since immunecomplexes and aggregated immunoglobulins are very large molecules whichmay not be able to enter the pores.

Suitable solid phase materials are well known in the art. Examplesinclude silica, polyacrylamide, polymethyl-methacrylate, polycarbonate,poly-acrylonitrile, polypropylene, polystyrene, latex beads and nylon.Commercial sources of suitable solid phase materials includeChromatoChem (Missoula, Mont.), Pharmacia Fine Chemicals (Uppsala,Sweden), and others.

Also, the C1q fragment or synthetic peptide is preferably covalentlyattached to the solid phase material. Methods and agents for affectingthis covalent attachment are well known in the art. Suitable agentsinclude carbodiimide, cyanoborohydride, diimidoesters, periodate,alkylhalides, succinimides, dimethylpimelimidate and dimaleimides SeeBlait, A. H., and Ghose, T. I., J. Immunol. Methods, 59:129 (1983);Blair, A. H., and Ghose, T. I., Cancer Res., 41:2700 (1981); Gauthier,et al., J. Expr. Med., 156:766-777 (1982)!.

The C1q fragment or synthetic peptide is also preferably attached to thesolid phase material by means of a spacer arm. The purpose of the spacerarm is to allow the fragment or peptide to be far enough away from thesurface of the solid phase so that it can interact with the immunecomplexes and aggregated immunoglobulins which are very large molecules.

Suitable spacer arms include aliphatic chains which terminate in afunctional group such as amino, carboxyl, thiol, hydroxyl, aldehyde, ormaleimido, which is active in a coupling reaction. The spacer arm may belocated on the solid phase or on the C1q fragment or synthetic peptideor, preferably, there is a spacer arm on both. If the spacer arm islocated on the C1q fragment or synthetic peptide, it is preferably apeptide containing less than ten amino acids, preferably two to threeamino acids.

The invention also comprises a method of detecting or quantitatingimmune complexes comprising contacting the immune complexes with the C1qfragment or the synthetic peptide so that the immune complexes bind tothe fragment or the peptide. The C1q fragment or synthetic peptide maybe added directly to fluids containing the immune complexes or may beattached to a solid phase of the types, and in the ways, describedabove.

The immune complexes can be detected or quantified using a labeledcomponent that binds to the immune complexes or to the C1q fragment orsynthetic peptide. For instance, labeled antibody to immunoglobulincould be used. The labels useful in the invention are those known in theart such as I¹²⁵, biotin, enzymes, fluorophores, bioluminescent labelsand chemiluminescent labels. Methods of binding and detecting theselabels are standard techniques known to those skilled in the art.

The immune complexes can be detected or quantitated using conventionalimmunoassay techniques. Such techniques include agglutination,radioimmunoassay, enzyme immunoassays and fluorescence assays. Enzymeimmunoassays (EIA) are preferred since they provide a means forsensitive quantitation of levels of immune complexes. The specificconcentrations, the temperature and time of incubation, as well as otherassay conditions, can be varied in whatever immunoassay is employeddepending on such factors as the concentration of the immune complexesor aggregated immunoglobulins in the sample, the nature of the sampleand the like. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination whileemploying routine experimentation.

Since body fluids from mammals normally contain immune complexes,comparison of the levels of immune complexes in a test sample from amammal will have to be made to the levels found in normals to identifylevels of immune complexes indicative of a disease state.

A test kit for detecting or quantitating immune complexes is also partof the invention. The kit is a packaged combination of one or morecontainers holding reagents useful in performing the immunoassays of theinvention.

The kit will comprise a container of the C1q fragment or a container ofthe synthetic peptide. The C1q fragment or synthetic peptide may be insolution or attached to a solid phase. The solid phases are the typesdescribed above, and the C1q fragment or synthetic peptide is attachedas described above.

The kit may further comprise a container holding the above-describedlabeled component that reacts with either the immune complexes or theC1q fragment or synthetic peptide. Finally, the kit may also containother materials which are known in the art and which may be desirablefrom a commercial and user standpoint. Such materials include buffers,enzyme substrates, diluents, standards, etc.

The C1q fragment or synthetic peptide can also be used as ananti-inflammatory drug. Inflammation in some diseases, such asrheumatoid arthritis, has been associated with the deposition of immunecomplexes in tissues and the activation of the complement cascade. It isthe binding of C1q to immune complexes deposited in the tissues whichinitiates the complement cascade, and the action of the complementcomponents, alone or concurrently with other biologic molecules,ultimately leads to tissue damage.

Accordingly, a C1q fragment or synthetic peptide according to theinvention can be administered to a mammal suffering from inflammationmediated by the classical complement pathway to inhibit the binding ofimmune complexes by C1q and, thereby, reduce or prevent tissue damageand further inflammation. The C1q fragments or synthetic peptides may beinjected at the site of inflammation in the mammal or may beadministered systemically (e.g., intravenously). The advantage of such atherapeutic approach is that small peptides are less likely to illicitan immune response which would render the drug inactive.

Xenotransplants are typically rejected within minutes to hours inuntreated recipients. This early rejection is called hyperacuterejection and results from recipients having preexisting xenoreactivenatural antibodies which bind to antigens of the endothelial cells ofthe donor organ, thereby activating the complement cascade. Thisultimately results in the formation of a membrane attack complex whichactivates the endothelial cells or penetrates the cell membrane,resulting in cell death and necrosis of the xenograft. The activation ofendothelial cells is devastating, resulting in thrombosis, inflammationand rejection of the transplanted tissue.

If hyperacute rejection is blocked and the organ lasts a few dayswithout immunosuppresive agents, the rejection which may occur withoutT-cell participation would be termed delayed xenograft rejection. Manyof the proinflammatory changes associated with delayed xenograftrejection are initiated by xenoantibody-mediated endothelial depositionof the complement components proximal to C3 (e.g., C1q). Blocking ofdelayed xenograft rejection would most likely lead to a rejectionsimilar to that seen with allografts.

The C1q fragments and synthetic peptides of the invention have beenfound to significantly prolong xenograft survival. For instance, in arat model of delayed xenograft rejection, xenograft survival wasprolonged from about one to three days in controls to up to about eightdays in treated animals.

Many individuals are not good candidates for allografts since theypossess polyreactive antibodies. These individuals reject transplantedallografts by an antibody-mediated mechanism which is analogous to thatseen in xenograft rejection, but occuring in "slow motion." Accordingly,it is anticipated that the C1q fragments and synthetic peptides of theinvention can be used to prolong survival of allografts in suchindividuals.

It has also been found that other peptides that inhibit the binding ofC1q to immunoglobulin may be used to treat inflammation and to prolonggraft survival. For instance, a Protein A fragment or a syntheticpeptide comprising the following sequence:

    Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile    1               5                   10    Leu His                      SEQ ID NO:8!

or variants thereof capable of binding immunoglobulin, may be used. SEQID NO:8 is from that portion of Protein A which binds to immunoglobulin.The binding of Protein A to immunoglobulin occurs near the C1q bindingsite, perhaps overlapping it, so the binding of the Protein A fragmentsand peptides to immunoglobulin hinders or prevents the binding of C1q.

Methods of administering peptides for therapeutic uses are well known.Effective dosage forms, modes of administration and dosage amounts maybe determined empirically, and making such determinations is within theskill of the art. It is understood by those skilled in the art that thedosage amount will vary with the particular peptide or fragmentemployed, the severity of the condition, the route of administration,the rate of excretion, the duration of the treatment, the identity ofany other drugs being administered to the mammal, the age, size andspecies of the mammal, and like factors well known in the medical andveterinary arts. In general, a suitable daily dose of a compound of thepresent invention will be that amount which is the lowest dose effectiveto produce a therapeutic effect. However, the total daily dose will bedetermined by an attending physician or veterinarian within the scope ofsound medical judgment. If desired, the daily dose may be administeredin multiple sub-doses, administered separately at appropriate intervalsthroughout the day.

For the above therapeutic uses, the fragments or peptides may be used assuch or may be bound to a macromolecule suitable for in vivo use. Suchmacromolecules include dextran and hetastarch.

Finally, the invention provides a binding material for removing immunecomplexes or aggregated immunoglobulins from a fluid. The materialcomprises plural binding peptides, the peptides being characterized inthat a plurality of them selectively binds immune complexes oraggregated immunoglobulins in the presence of monomeric immunoglobulin.Preferably, the binding peptides are the C1q fragments of the inventionor synthetic peptides comprising the sequence

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr     1               5                   10                      SEQ ID NO 2!

or combinations of the two.

Individual binding peptides have a low affinity for immunoglobulin.Accordingly, the plurality of binding peptides must be held insufficient proximity to each other so that multiple points of attachmentto the immune complex or aggregated immunoglobulins can be made and afunctional affinity is formed. This can be accomplished by formingoligomers of the binding peptides or by attaching the binding peptidesto a solid phase at an effective density as described above for the C1qfragments and synthetic peptides. The methods of preparation, andproperties, of the resulting oligomers and solid phase materials are asdescribed above for the C1q fragments and synthetic peptides. Also, theoligomers of C1q fragments or synthetic peptides and the solid phasematerials having C1q fragments or synthetic peptides attached to them atan effective density that are described above are examples of bindingmaterials according to the invention.

Immune complexes and aggregated immunoglobulins can be removed fromfluids containing them by contacting the fluids with the bindingmaterial, and then separating the fluid from the binding material. Thefluid is simply contacted with the binding material. Such contact can beeffected by passing the fluid through a device containing the bindingmaterial so that the fluid may contact the binding material.Alternatively, the fluid may be incubated statically with the bindingmaterial in the device. The duration of the contact is not bound tocritical limits although it should, of course, be sufficient to allowaggregated immunoglobulin or immune complexes to be bound by the bindingpeptides. The binding material of the invention is especiallywell-adapted to be used to remove aggregated immunoglobulins or immunecomplexes from fluids containing monomeric immunoglobulin such as thoselisted above.

The invention also comprises a device for removing immune complexes oraggregated immunoglobulins from a fluid. The device comprises thebinding material and a means for encasing the material so that the fluidcan be contacted with it. Binding materials having binding peptidesattached to a solid phase are preferred.

The encasing means may be a plastic bag, a column, a test tube, plastictubing, encasing means like those used on plasmapheresis devices, andother suitable encasing means. The encasing means should be made of amaterial which is not harmful to the fluid to be placed in the device.

Thus, the device may be a typical plasmapheresis device in which thesolid phase is a membranous surface or hollow fibers to which thebinding peptides are attached. The device may also be a column packedwith beads or any suitable solid phase having the binding peptidesattached to it. The device may be a test tube filled with beads to whichthe binding peptide is attached.

Other devices are also possible. For instance, the binding material maybe beads (such as silica beads) to which the binding peptides areattached at a suitable density to selectively remove immune complexes oraggregated immunoglobulins from fluids containing monomericimmunoglobulin. The means for encasing the beads may be a plastic bag ofe.g., the type used for transfusions. The fluid containing the immunecomplexes or aggregated immunoglobulins is mixed with the beads in thebag and incubated for a time, and at a temperature, sufficient to allowthe immune complexes or aggregated immunoglobulins to bind to thebinding peptides. Then the beads are allowed to settle (or may becentrifuged), and the fluid, from which immune complexes have beenremoved, is decanted.

For therapeutic uses, a solid phase binding material can be encasedonline in an extracorporeal device through which whole blood or plasmacan be circulated dynamically so that the immune complexes containedtherein are bound and removed from the blood or plasma. An alternativewould be to statically incubate the whole blood or plasma in a devicesuch as the plastic bag device described above. In either case, fluidscan be returned to the body after the incubation or passage is complete,negating the need for blood replacement therapy.

A device intended for therapeutic use may also include appropriatetubing for connecting it to a patient and a pump to aid the passage ofthe fluid through the device and back into the patient and to preventair from entering the system. The device must be sterilized fortherapeutic use, and sterilization may be accomplished in conventionalways such as purging with ethylene oxide or by irradiating the device.

EXAMPLES

Unless otherwise indicated, the chemicals used in the following Exampleswere obtained from Sigma Chemical Co., St. Louis, Mo.

Example 1 Peptide Synthesis

A. Peptide Design and Structure

X-ray crystal structural data for Protein A Fragment B indicated thattwo coplanar α-helical segments with the proper contact residues (seeBackground) could bind immunoglobulin Fc with a high affinity.Deisenhofer et al., Hoppe-Seyler's Z. Physiol. Chem. Bd., 359, S. 975-85(1978); Burton, Molec. Immunol., 25, 1175-81 (1988); Langone, Adv.Immunol., 32, 157-252 (1982). As discussed above, C1q also binds IgG Fc.Thus, the question was posed whether C1q bound IgG Fc using structuressimilar to those found in Protein A Fragment B. Unfortunately,structural information regarding the C1q molecule was limited, andinformation concerning the C1q binding site for immunoglobulin wasnonexistent.

However, the amino acid sequences of the entire A and B chains and apartial sequence for the C chain of C1q were available. Reid,Biochemical J., 179, 367-71 (1978); Reid, et al., Biochemical J., 203,559-69 (1982). These sequences were analyzed by the secondary structureprediction programs of Garnier et al., J. Molec. Biol., 120, 97-120(1978) and Chou and Fasman, Adv. Enzym., 47, 45-146 (1978). Of thepredicted alpha helical regions, one helical region in the C1q B chainglobular head region spanned 12 residues, suggesting a high probabilitythat the helix prediction was accurate.

The predicted helical segment of C1q B chain (residues 189-200) was thencompared to one Protein A Fragment B helix (residues 142-153), andhomology was observed (see FIG. 1). Four amino acids were exact matches,while three additional residues showed conservative substitutions. Outof three possible contact residues on the Protein A helix, C1q possessedtwo similar residues. Further analysis of the two sequences was achievedthrough the use of helical wheel diagrams (FIG. 2). These diagramsshowed that the pattern of hydrophobic and hydrophilic residues observedfor the Protein A helix known to contact immunoglobulin was similar tothat of the predicted C1q helix.

Therefore, the 12-residue sequence of C1q having the predicted helicalstructure was synthesized. Two Leu residues were added to thecarboxyl-terminus as spacing residues to separate the potentially activeresidues from a future solid phase matrix. A Cys residue was also addedto the carboxyl-terminus to allow for coupling to solid phases usingcertain coupling procedures. The final amino acid sequence, designatedCBP2, is presented in FIG. 1.

B. Synthesis

The CBP2 sequence was submitted to Biosearch Inc. (San Rapheal, Calif.)for custom peptide synthesis using the standard Merrifield solid phasesynthesis approach. Merrifield, in Chem. Polypeptides, pp. 335-61(Katsoyannis and Panayotis eds., Plenum, New York, N.Y. 1973).Approximately 250 mg of the peptide in lyophilized form were obtained.

Biosearch's quality control information was based on amino acid analysisand analytical reverse phase HPLC. The chromatograph provided byBiosearch showed that the initial purity of the peptide wasapproximately 40-50%. Biosearch did not provide the results of the aminoacid analysis, but they stated that the composition of the peptide hadto be within +/-20% of the calculated values for each amino acid of thepeptide in order to pass their quality control. Therefore, the CBP2peptide should have had the correct amino acid sequence.

C. Purification

In addition to the purification performed by Biosearch, the peptide wasfurther purified by gel filtration chromatography. The lyophilized CBP2was not soluble in aqueous solvents and required 50% (v/v)N,N-dimethylformamide (DMF) (J. T. Baker Chemicals, Phillipsburg, N.J.)to completely dissolve. Due to the high hydrophobic character of thesolvent, Sephadex G25 (Pharmacia, Piscataway, N.J.) was used for thechromatography. A column of dimensions 0.7×40 cm was poured andequilibrated with 1% (v/v) DMF in distilled water (dH₂ O). A 2 mg/mlsample of CBP2 was prepared in 50% (v/v) DMF, and 1 ml of the sample wasloaded on the column and eluted with 1% DMF at a flow rate of 1 ml/15min. One ml fractions were collected.

The column fractions were assayed for peptide by the Bicinchoninic Acid(BCA) protein assay (Pierce Chemical Co., Rockford, Ill.) according tothe manufacturer's instructions. Briefly, duplicate 30 μl aliquots fromeach fraction were placed in the wells of a microtiter plate, followedby the addition of 300 μl of BCA protein reagent. In addition to thecolumn fractions, samples ranging from 1 mg/ml to 0.1 μg/ml Val-Gly-Glypeptide (Sigma Chemical Co., St. Louis, Mo.) were also added to themicrotiter plate to prepare a standard curve. The plate was incubated at37° C. for 1 hour, and the resultant colored product measured using aTiterTek Multiskan plate reader (Flow Laboratories, McLean, Va.) at 560nm. The absorbances were then plotted against fraction number togenerate the chromatogram.

A 1% (v/v) DMF sample was also tested in the BCA assay as a control. DMFwas shown by these experiments not to interfere with the detection ofthe peptides in the column fractions.

A 1.2 fold increase in peptide purity with a 41% yield of peptide wasobtained. A representative chromatograph for CBP2 is shown in FIG. 3.The profile showed a symmetrical major peak at 250 minutes and severalminor peaks representing contaminants. It should also be noted thatpurified CBP2 was readily soluble in 1% (V/V) DMF, indicating that itmay be the impurities in lyophilized CBP2 that could only be dissolvedin 50% DMF.

In addition to the standard BCA assay for peptides, the absorbanceprofile for CBP2 migrating in the Sephadex G25 column was measured at230 nm. The absorbance profile shown in FIG. 3 confirms the results ofthe BCA assay in that the CBP2 peak had a retention time of 250 minutes.It is important to note that the UV absorbance profile at 230 nm is mostsensitive to amide bonds. Since the CBP2 peak was the major source ofabsorbing material, the remaining impurities may not contain amide bondsand, therefore, may not be peptides.

The CBP2 retention time in the Sephadex G25 column suggested a largermolecular weight than the calculated molecular weight based on the aminoacid sequence of the peptide. Therefore, under the same conditions,samples of glucagon, LHRH, and a mixture of glucagon and LHRH wereloaded and eluted from the Sephadex G25 column. Glucagon (3550 daltons)had a retention time of 247 minutes and LHRH (1182.33 daltons) had aretention time of 277.4 minutes. CBP2 (1556.98 daltons) had a retentiontime similar to glucagon, suggesting that CBP2 migrated through theSephadex G25 column as an aggregate of at least two peptides.Concentration dependent aggregation of amphipathic peptides has beendocumented. DeGrado et al., J. Am. Chem. Soc., 103, 679-81 (1981);Taylor et al., Mol. Pharmacol., 22, 657-66 (1982); Moe and Kaiser,Biochem., 24, 1971-76 (1985).

Analytical C₁₈ reverse phase HPLC was used in order to assess the purityof the CBP2 isolated from the Sephadex G25 column. The conditions usedfor the chromatography were the same as those used by Biosearch.Briefly, 500 μg/ml of CBP2 in 1% (v/v) DMF in dH₂ O and 4 mg/mldithiothreitol (the peptide was incubated over night with the DTT inorder to reduce any disulfide bonds that may have formed) were passedover a Vydac (Hesperia, Calif.) C₁₈ analytical column (product #218TP54)with dimensions 4.6 mm×25 cm and using the following buffers andconditions. Buffer A: 0.05% (v/v) trifluoroacetic acid (TFA) in dH₂ O.Buffer B: 0.05% (v/v) TFA in acetonitrile. Column conditions: A 100 μlsample was injected, and 5% buffer B was maintained for 3 min at 1.7ml/min followed with a gradient of 5-100% buffer B over the next 20 min.The peptide was detected with a Beckman (Palo Alto, Calif.) UV variablewavelength detector at 230 nm.

It is important to note that CBP2 was treated with DTT overnight beforethe analysis in order to disrupt any disulfide bonds that may haveformed. FIG. 4A shows a representative chromatograph of the reducedisolated CBP2. CBP2 comprised 80% of the absorbing material, indicatingthat the Sephadex G25 isolated CBP2 was relatively pure. With exceptionof the peaks at 4.4 minutes, the remaining peaks were retained in thecolumn significantly longer than CBP2, suggesting that the impuritieswere of smaller molecular weight and/or were much more hydrophobic thanCBP2.

Since CBP2 did contain free cysteine residues, it was necessary todetermine the CBP2 profile in the absence of DTT to assess the extent ofdisulfide formation. In addition, K₃ Fe(CN)₆, which would favor theformation of disulfides, was added to the reduced CBP2 solution. Thisprocess was necessary to determine the retention time of the disulfideaggregated CBP2. FIG. 4B shows the CBP2 profile without the reduction ofdisulfides. A strong absorbance peak at 4.4 minutes possessing 28% ofthe total absorbance was the only significant difference between reducedand nonreduced CBP2 (compare FIGS. 4A and 4B). The addition of K₃Fe(CN)₆ to the reduced CBP2 solution and the subsequent analysis showedthat the peak at 4.4 minutes in FIG. 4B was due to disulfide aggregatedCBP2. In FIG. 4C, the addition of the oxidizing reagent caused adecrease in the reduced CBP2 peak with an accompanying increase in the4.4 minute peak which represented 68.7% of the total area. Therefore,the 4.4 minute peak was the disulfide aggregated CBP2.

It is important to note that in all three chromatographs in FIG. 4, CBP2comprises at least 80% of the total area, with the combination of thereduced and nonreduced CBP2 peaks representing approximately 90% of thetotal area. Based on the results of the three chromatographs, it wasconcluded that the purity of CBP2 was 80-90%.

D. CD Spectroscopic Analysis Of CBP2

Circular dichroism (CD) spectroscopy was performed on an Aviv 60DSspectropolarimeter. The sample consisted of 200 μl of 200 μg/ml CBP2dissolved in 10 mM sodium phosphate buffer, pH 7.0, at room temperature.A 1 mm path length was used.

Under these conditions, CBP2 exhibited a CD spectrum characteristic of arandom coil peptide. The lack of structure was expected for smallpeptides such as CBP2.

Example 2 Inhibition Studies

A. Titration of Rabbit Immunoglobulin Bound by Solid Phase Protein A

Protein A (recombinant Protein A from Repligen, Cambridge, Mass.)diluted to 1 μg/ml in coating buffer (0.1M NaHCO₃, pH 9.0) was incubatedin the wells of a microtiter plate (100 μl per well) for 2 hours at 37°C. Uncoated surfaces of the wells were blocked by adding 340 μl PBSC (9mM Na₂ HPO₄, 1 mM NaH₂ PO₄, pH 7.2, 154 mM NaCl, 1 mg/ml casein orovalbumin, and 0.01% thimerosal), incubating the plate 1 hour at 37° C.,and then washing the wells 3 times with PBSCT (PBSC plus 0.1% Tween 20(polyoxyethylene sorbitan monolaurate)). Dilutions of rabbitimmunoglobulin labeled with horseradish peroxidase (Ig-HRP) (ZymedLaboratories, Inc., South San Francisco, Calif.) in PBS (9 mM Na₂ HPO₄,1 mM NaH₂ PO₄, pH 7.2, 154 mM NaCl) were incubated in the wells (100 μlper well) for 2 hours at 37° C. followed by washing 3 times with PBS.OPD substrate buffer (0.8 mg/ml o-phenylenediamine, 0.1M Na₂ HPO₄, 0.05Msodium citrate, pH 5.5, plus 0.15% (v/v) H₂ O₂) was then added to thewells (100 μl/well), and the resultant colored product was measured witha Titertek Multiskan plate reader using a 414 nm filter.

B. CBP2 Inhibition of Solid Phase Adsorbed Protein A.

It had previously been reported that C1q and Protein A competeexclusively for IgG (Burton, Molec. Immunol., 22, 161-206 (1985);Stalinheim et al., Immunochem., 10, 501-507 (1973); Langone et al., J.Immunol., 121, 327-38 (1978); Lancet et al., Biochem. Biophys. Res.Commun., 85, 608-614 (1978)). Accordingly, CBP2 was tested forinhibition of the binding of Ig-HRP by Protein A.

The wells of a microtiter plate were coated with Protein A as describedabove in Part A. Ig-HRP diluted 1:5000 in PBS (the dilution determinedby the protein A titration experiment of Part A; original Ig-HRPconcentration of 3.3 mg/ml) was preincubated with various concentrationsof CBP2 for 1 hour at 37° C. before 100 μl of the incubation mixturewere added to each well of the microtiter plate. The CBP2/Ig-HRP mixturewas incubated with the solid phase adsorbed Protein A for 2 hours at 37°C. and then washed 3 times with PBS. OPD substrate buffer was then added(100 μl/well), and the resulting colored product was measured at 414 nmin a Titertek Multiskan plate reader. Absorbance readings were made whenthe control wells with no inhibitor had an absorbance of 1.0 or greater.

The results, which are shown in FIG. 5, show that CBP2 was able toinhibit Protein A binding of Ig-HRP by nearly 100%. The concentration at50% inhibition was approximately 1 μM which is a 1000-fold greaterconcentration than the 1 nM 50% inhibition concentration for solutionphase Protein A (see FIG. 5). Since the 50% inhibition concentration forProtein A is comparable to its dissociation constant, the 50% inhibitionconcentrations were considered a good measure of the binding affinity ofthe inhibitor.

C. Titration of Ig-HRP Bound by Solid Phase C1q.

C1q (gift of Dr. Lawrence Potempa, Immtech International Inc., Evanston,Ill.) diluted to 10 μg/ml in coating buffer was placed in the wells of amicrotiter plate (100 μl/well) and incubated for 2 hours at 25° C. Theuncoated surfaces of the wells were blocked by adding 340 μl PBSC andincubating the plate for 1 hour at 25° C., followed by washing 3 timeswith PBSC. Dilutions of Ig-HRP in C1q buffer (50 mM Tris, pH 7.2, 27 mMNaCl, 1 mM CaCl₂, 0.01% (wt/v) Thimerosal) were incubated in the wells(100 μl/well) for 1 hour at 25° C. followed by washing 3 times with C1qbuffer. OPD substrate buffer was then added (100 μl/well), and thecolored product was measured at 414 nm in a Titertek Multiskan platereader.

D. CBP2 Inhibition of Solid Phase C1q.

C1q was coated onto the surface of the wells of a microtiter plate asdescribed in Part C. Ig-HRP diluted 1:100 (the dilution determined fromthe C1q titration experiment described above in Part C) in C1q bufferwas preincubated with various concentrations of CBP2 for 1 hour at 25°C. The Ig-HRP/CBP2 mixture was then added to the microtiter plate (100μl/well), and the plate was incubated for 1 hour at 25° C., followed by3 washes with C1q buffer. OPD substrate buffer (100 μl/well) was thenadded and the colored product measured at 414 nm in a Titertek Mutiskanplate reader.

The resulting inhibition curve is presented in FIG. 6. The 50%inhibition concentration was approximately 10 μM, which was an order ofmagnitude higher than the 50% inhibition concentration for CBP2inhibiting Protein A. The 50% inhibition concentration for liquid phaseC1q inhibiting solid phase C1q was approximately 10 nM or 1000 fold lessthan the CBP2 concentration (see FIG. 6).

E. Control Experiments For CBP2 Inhibition Of Protein A And C1q

In order to conclude that CBP2 was binding immunoglobulin and notcausing an inhibition in the assay system through a nonspecificinteraction, a series of control experiments were performed.

1. The effects of CBP2 on the HRP enzyme were investigated. Ig-HRP at a1:100 dilution in PBS or C1q buffer was incubated with 50 μM CBP2 or anequal amount of 1%(v/v) DMF for 3 hours at 37° C. Aliquots of 100 μlwere placed in the wells of a microtiter plate and an equal volume ofOPD substrate buffer was added, and the colored product was measured at414 nm. Both the CBP2 and Ig-HRP were at the highest concentrationstested in the inhibition assay. The sample which contained both peptideand Ig-HRP did not differ significantly from the control samplecontaining the Ig-HRP alone. Therefore CBP2 did not inhibit the HRPenzyme.

2. Protein A at 1 μg/ml or C1q at 10 μg/ml diluted in coating buffer wasincubated in the wells of a microtiter plate (100 μl/well) for 2 hoursat 37° C. or 25° C., respectively. The wells were blocked in the samefashion as described above for each assay. Various concentrations ofCBP2 diluted in PBS or C1q buffer were incubated in the wells (100μl/well) for 2 hours at 37° C. or 1 hour at 25° C., respectively,followed by washing the wells 3 times with either PBS or C1q buffer.Ig-HRP was then added at the appropriate dilution in either PBS or C1qbuffer and incubated under the appropriate conditions, followed bywashing 3 times with PBS or C1q buffer. OPD substrate buffer was thenadded (100 μl/well), and the colored product was measured at 414 nm asdescribed above.

3. Excess protein (1 mg/ml casein) was added to the buffers used todilute the Ig-HRP/CBP mixture.

Controls 2 and 3 tested for nonspecific protein-protein orprotein-peptide interactions. If there were such interactions, thenthere would be a loss of CBP2's inhibitory activity in test 3, and nosuch loss was observed. In test 2, any loss in CBP2's inhibitoryactivity would indicate that CBP2 was blocking the solid phase Protein Aor C1q binding sites and not interacting with Ig-HRP. No loss ofinhibitory activity was detected.

4. Equal molar concentrations of luteinizing hormone releasing hormone(LHRH) (Beckman Instruments, Palo Alto, Calif.) or variousconcentrations of Protein A were substituted for CBP2 in the inhibitionassay described in Part B. In the case of the C1q inhibitionexperiments, LHRH and C1q were substituted for CBP2 as a negative and apositive control, respectively. The use of LHRH also tested whether theinhibition of Protein A or C1q by CBP2 was due to CBP2 specifically, orsimply due to nonspecific effects attributable to a small peptide. Theresults are shown in FIGS. 5 and 6. As indicated in these figures, LHRHdemonstrated no Protein A or C1q inhibitory activity over the sameconcentration range examined for CBP2, and both Protein A and C1q wereinhibitory, as expected.

5. Concentrations of Ig-HRP required for the C1q assays were 50 foldhigher than the concentration used for Protein A assays. The reason forthe higher concentration is that only a small fraction of Ig-HRP existedas complexes that could be bound by C1q. This was demonstrated byfractionating the Ig-HRP as follows. A 40 μl sample of Ig-HRP was loadedonto a TSK SW 4000 sizing HPLC column (Beckman/Altex TSK 4000 SW, 7.5mm×30 cm). Only the high molecular mass eluted fractions exhibited anyC1q binding activity as determined by a C1q binding assay performed asdescribed above. The results of this assay indicated that the C1qbinding material was a fraction having molecular weight of approximately1,000 kdal, indicating a complex of immunoglobulins and HRP enzymes, andnot a monomeric immunoglobulin plus HRP enzyme.

6. With respect to C1q, nonspecific ionic interactions could not beexcluded since the C1q buffer was half-physiological ionic strength. Theionic strength of the C1q buffer could not be changed since C1q bindingof immunoglobulins is partially mediated by ionic interactions. However,based on the Protein A inhibitory activity of CBP2 in physiologicalionic strengths, it was a reasonable assumption that CBP2 inhibition wasnot primarily the result of nonspecific ionic interactions.

7. Proteins when adsorbed to the polystyrene wells of a microtiter platemay partially lose their native conformation. Therefore, CBP2 was alsoscreened for any interactions with solution phase Protein A. Protein Awas incubated in the wells of a microtiter plate (100 μl/well) for 2hours at 37° C. at 1 μg/ml in coating buffer, and the wells blocked asdescribed above. Protein A at a constant concentration (corresponding toeither 100 times the 50% inhibition concentration, the 50% inhibitionconcentration, or 10 fold less than the 50% inhibition concentration)was preincubated with CBP2 and Ig-HRP in PBS for 1 hour at 37° C. TheProtein A/CBP2/Ig-HRP mixture was then incubated in the wells of themicrotiter plate (100 μl/well) for 2 hours at 37° C. and washed 3 timeswith PBS. OPD substrate buffer was then added (100 μl/well), and theresultant colored product was measured at 414 nm as before.

At Protein A concentrations at or above the 50% inhibitionconcentration, there was a linear, additive response, indicating nointeraction between CBP2 and Protein A. At Protein A concentrationsbelow the 50% inhibition concentration, the curve is analogous to theCBP2 inhibition curve in the absence of solution phase Protein A. Thisalso suggests that CBP2 and Protein A are not interacting.

8. Since the CBP2 peptide has a free cysteine residue, the effects ofDTT on the inhibition assay were examined. DTT was added to the buffercontaining CBP2 (final DTT concentration 4 mg/ml), and the CBP2 wasincubated with the DTT for 2 hours at 37° C. to ensure that the cysteineresidues of CBP2 were reduced. Then the inhibition assay using solidphase Protein A was performed as described above.

The addition of DTT to the CBP2 solution had no effect on the inhibitoryactivity of CBP2. These data indicate that CBP2 does not inhibit byforming disulfide interaction with Protein A or Ig-HRP and thatdisulfide-mediated CBP2 aggregates did not have a significant influenceon CBP2's ability to inhibit the binding of Ig-HRP to Protein A.

F. Conclusions

Based on the inhibition data in conjunction with the above controls, itcan be concluded that CBP2 inhibits C1q and Protein A binding of Ig-HRPthrough a binding interaction with solution phase Ig-HRP. The resultsindicate that CBP2 binds immunoglobulin in a specific manner.

Example 3 HiPAC™ LTQ Column

A HiPAC™ LTQ (ChromatoChem, Missoula, Mont.) activated aldehyde columnof dimensions 7.4 mm×1.9 cm was equilibrated with 6 ml of immobilizationbuffer (0.1M sodium citrate, pH 5.5). This column is composed of silicabeads to which a long carbon chain spacer arm is attached. A ligandcoupling solution containing 500 μg CBP2 and 20 mg/ml sodiumcyanoborohydride in immobilization buffer was prepared. This wascontinuously circulated through the column for 5 min at 25° C. at a flowrate of 1-2 ml/min. The coupling solution was then eluted from thecolumn and collected. The column was subsequently washed with 6 mlimmobilization buffer which was also collected. The column was thenwashed with 6 ml of 2% (v/v) acetic acid and equilibrated with 6 ml C1qbuffer.

The coupling procedure linked CBP2 to the solid phase support throughthe N-terminal amino group, leaving the C-terminal cysteine's sulfhydrylavailable as a marker. The Ellman's reagent assay (Ellman, Arch.Biochem. Biophys., 82, 70-77 (1959)) was, therefore, used to measure theamount of CBP2 bound to the column since Ellman's reagent(5,5-dithio-bis-(2-nitrobenzoic acid)) reacts with free sulfhydrylgroups.

To perform the Ellman's reagent assay, the column was first washed witha 4 mg/ml solution of DTT in order remove any peptides linked to thesolid phase by disulfide bonds. Then the column was washed with C1qbuffer until the eluate showed no reactivity with Ellman's reagent. Thecolumn was then equilibrated with 6 ml of 0.1N Na₂ HPO₄, pH 8.0. Next, 1ml of a solution of 400 μg/ml Ellman's reagent in 0.1N Na₂ HPO₄, pH 8.0,was continuously passed over the column for 15 min. The Ellman's reagentsolution was eluted from the column, collected, and the column washedwith 6 ml of 0.1N Na₂ HPO₄, pH 8.0. One ml of a 4 mg/ml solution of DTTwas passed over the column, and the eluate was collected. The column wasthen washed with 6 ml C1q buffer and the eluates collected. Theresulting colored products were measured at 412 nm.

The moles of CBP2 coupled to the column were proportional to the molesof Ellman's reagent reacting with the column (i.e., the amount ofEllman's reagent eluted with the DTT) which was calculated using a molarextinction coefficient of 1.36×10⁴ /cm·M for the free thionitrobenzoicacid. The assay of the column indicated that 84% of the CBP2 added tothe column was coupled to the solid phase, or approximately 420 μg CBP2.

It should be noted that attempts to measure CBP2 in eluted fractionsusing various techniques (BCA assay, UV spectroscopy,2,4,6-trinitrobenzene sulfonic acid and the Ellman's reagent assay) wereunsuccessful because the cyanohydride reagent interferred with thoseassays.

Two control columns were prepared along with the CBP2 column. LHRH wascoupled to the matrix in the same fashion as CBP2 and at the sameconcentration as CBP2 as determined by UV spectroscopy. A similarpercentage (78%) of LHRH was coupled to the column by reductiveamination as with CBP2 (84%). Another column was treated in the samefashion as the CBP2 and LHRH columns, but no peptide or protein wascoupled ("No peptide" column), thus allowing the study of nonspecificbinding effects in the following experiments.

Example 4 PAP Binding To The HiPAC™ LTO-CBP2 Column.

A CBP2 column prepared as described in Example 3 was equilibrated with 3ml C1q buffer. Immune complexes composed of rabbit anti-horseradishperoxidase and horseradish peroxidase (PAP) (purchased from OrganonTeknika/Cappel, West Chester, Pa.) at a concentration of 20 μg/ml in C1qbuffer (a total volume of 1 ml) were loaded onto the column andincubated for 15 min at 25° C. with continuous circulation through thecolumn. Unbound PAP complexes were eluted with 12 ml C1q buffer (at aflow rate of 0.33 ml/min, collecting 1 ml fractions) followed by 3 ml of2% (v/v) acetic acid to elute bound PAP.

A. Peroxidase Assays

The column fractions were assayed for peroxidase activity by placingduplicate 20 μl aliquots into the wells of a microtiter plate and adding100 μl of OPD substrate buffer. The absorbances were measured at 414 nmin a Titertek Multiskan plate reader. The absorbances were then plottedagainst fraction number to generate the chromatograph. In addition tothe samples, a standard curve was prepared, and a linear regressionanalysis was performed. From the standard curve the concentration of PAPin each fraction was calculated.

The resulting profile is presented in FIG. 7. The elution profile showstwo major peaks. The first peak was material that did not bind to thecolumn and which was readily eluted with C1q buffer. This materialappeared to make up the larger fraction of the PAP loaded on the column.In fact, using the standard curve and linear regression analysis, theflow-through peak was calculated to contain approximated 90-95% of thetotal PAP. The second peak was the material bound by the column andeluted with the acetic acid. The bound material was only 5-10% of thetotal PAP loaded on the column.

Although the efficiency of the column appeared to be low, the column didbind immune complexes. Therefore, it was determined whether the columnbound immune complexes specifically and in a CBP2-dependent manner.

One ml containing PAP complexes at a concentration of 20 μg/ml andmonomeric rabbit IgG at a concentration of 80 μg/ml in C1q buffer wasloaded on a CBP2 column prepared as described in Example 3, that hadbeen equilibrated with 3 ml of C1q buffer. The PAP/IgG mixture wascirculated through the column continuously for 15 min and then elutedwith 12 ml C1q buffer at a flow rate of 0.33 ml/min. Material bound tothe column was eluted with 3 ml of 2% (v/v) acetic acid followed by 6 mlof C1q buffer. (The flow rates and fractions were the same as for PAPalone.) The fractions were assayed for the presence of peroxidaseactivity in the same fashion as described above and the concentration ofPAP was calculated from the standard curve.

Once again two peaks were observed (FIG. 8). The first peak representedthe flow-through, and contained 95% of the total PAP loaded on thecolumn. The amount of material bound to the column in the second peakalso remained unchanged at approximately 5%. Since the elution profilesof PAP, both with and without monomeric IgG, were not different withrespect to the quantities of material in each peak and the retentiontimes of the peaks, it appeared that the column specifically boundimmune complexes.

B. Enzyme Immunoassay For IgG

PAP complexes were loaded onto a CBP2 column and eluted from it asdescribed in Part A. Also, monomeric IgG at a concentration of 80 μg/mlin 1 ml of C1q buffer was loaded onto a CBP2 column and eluted from itin the same manner.

The fractions were then assayed for immunoglobulin by enzyme immunoassayas follows. Duplicate 50 μl aliquots of each fraction were placed in thewells of a microtiter plate. Fifty μl of coating buffer were then added,and the plate was incubated at 37° C. for 2 hours. The uncoated surfacesof the wells were then blocked by adding 340 μl of PBSC, followed byincubation of the plate for 1 hour at 37° C. The wells were washed 3times with PBSCT, and a 1:2000 dilution of goat anti-rabbit IgG that waslabeled with biotin (anti-rabbit IgG) (Vector Laboratories, Inc.,Burlingame, Calif.) was added (100 μl/well). The anti-rabbit IgG wasincubated in the wells for 2 hours at 25° C., and the wells were washed3 times with PBSCT. Next, a 1:2000 dilution ofstreptavidin-β-galactosidase (BRL-Life Technologies, Gaithersberg, Md.)in PBSCT was added to the wells (1000 μl/well), the plates wereincubated for 2 hours at 25° C. and washed 3 times with PBSCT.Fluorogenic substrate in buffer (5 mg/ml of4-methylumbelliferyl-β-D-galactoside in DMF diluted, 1:50 in 0.01Msodium phosphate buffer, pH 7.5, containing 0.1M NaCl and 1 mM MgCl₂)was added to the wells (100 μl/well), and the resulting fluorescence wasmeasured with a Dynatech MicroFluor plate reader (Dynatech, Alexandria,Va.) using 365 nm as the excitation wavelength and 450 mn as theemission wavelength.

In addition to the samples, standard curves of IgG and PAP dilutionswere prepared, and a linear regression analysis was performed. Thestandards demonstrated a linear response in fluorescence to PAP and IgGconcentration as confirmed by the linear regression analysis. From thestandard curves, the concentration of PAP or IgG in each fraction wascalculated.

The linear regression analysis showed that 44% of the PAP loaded on thecolumn were bound to the column. The amount of monomeric IgG bound tothe column was 4.4% of the total IgG loaded on the column. See Table 1below. These results show that the column was not binding a significantamount of monomeric IgG and was specifically binding immune complexes.

Clearly, there was a large discrepancy between the results of theperoxidase assay (about 5% binding of immune complexes) and the enzymeimmunoassay for immunoglobulin (about 44% binding of immune complexes).Closer examination of the kinetics of the two enzymatic reactionsrevealed that the peroxidase assay reached its maximal absorbance in5-10 minutes on average, while the streptavidin-β-galactosidase assayreached its maximal fluorescence in 25-30 minutes. Therefore, althoughthe two assays both gave linear responses to incremental increases inconcentration of PAP, the enzyme immunoassay measured the relativequantities of PAP more accurately and sensitively due to the longerincubation time.

As a result of the comparison of the results of the peroxidase assay andenzyme immunoassay, elution profiles were reassessed using the enzymeimmunoassay. Also, PAP complexes were loaded onto and eluted from thecontrol LHRH and No peptide columns in the same manner as describedabove for the CBP2 column and assayed using the enzyme immunoassay. Theresults are shown in Table 1 below.

                  TABLE 1    ______________________________________    Percent Bound to Column    Sample   CBP2 Column                        LRHR Column No Peptide Column    ______________________________________    PAP      44         4.8         8    Monomeric             4.4        N.D..sup.a  N.D..sup.a    Rabbit IgG    ______________________________________     .sup.a Not Determined

The immune complex binding efficiency of the CBP2 column as shown inTable 1 was much higher than had been previously calculated using theperoxidase assay. The results also indicated that the columnspecifically bound immune complexes. There was very little binding ofthe immune complexes to the control LHRH and No peptide columns, showingthat the binding of immune complexes was not due to nonspecific bindingeffects.

C. Elution With CBP2

If PAP binding was a specific interaction between the solid phase CBP2and the immunoglobulins of PAP, then PAP bound by the column should beeluted using a solution of CBP2, and a solution of a control peptide(LHRH) should cause no elution of bound PAP. This was exactly what wasfound by the following experiment.

A 1 ml sample of 20 μg/ml PAP was loaded on an equilibrated CBP2 column(prepared as described in Example 3) and allowed to circulatecontinuously for 15 min over the column. The column was then washed with12 ml C1q buffer, followed by 3 ml of 336.2 μg/ml CBP2 (this CBP2concentration was 20 times the concentration of CBP2 needed to give 50%inhibition Ig-HRP binding to C1q) diluted in C1q buffer. An additional 6ml of C1q buffer was used to wash the column followed by 3 ml of 2%(v/v) acetic acid and then with 6 ml of C1q buffer to completely washthe column. Fractions were collected and assayed for the presence ofperoxidase activity as described above in Part A. A control column wasrun as described above, except that 3 ml of LHRH at an equimolarconcentration as the CBP2 solution, was substituted for the CBP2 wash.

As shown in FIG. 9A, the normal flow-through peak was observed, followedby a small but significant PAP peak which was eluted from the columnwith CBP2. The remaining PAP were eluted by the acetic acid/C1q bufferwash. When the experiment was repeated with LHRH at an equimolarconcentration, there was no significant elution of PAP due to the LHRHwash (see FIG. 9B). These results show that PAP binding to the columnwas dependent on CBP2.

D. Controls

1. The low efficiency of PAP elution by the CBP2 solution could havebeen explained by either: 1) the presence of inactive CBP2 aggregateseffectively lowering the free CBP2 concentration; or 2) because the PAPelution occurred under nonequilibrium conditions. Under nonequilibriumconditions, the CBP2 peptide would not adequately compete with the solidphase CBP2 for binding sites on the PAP since a high affinityinteraction (i.e., a functional affinity) between the solid phase CBP2and the bound PAP had been established.

To address the question of nonequilibrium conditions, a 1 ml sample of20 μg/ml PAP plus 200 μg/ml CBP2 (0.1 mM; a ten times greaterconcentration than that required to give 50% inhibition of Ig-HRPbinding to C1q) or 152 μg/ml LHRH (0.1 mM) was incubated for 1 hour at25° C. and then loaded on a pre-equilibrated CBP2 column and allowed topass continuously over the column for 15 min. The sample was then elutedwith 21 ml C1q buffer followed by 3 ml of 2% (v/v) acetic acid and then6 ml C1q buffer. The column fractions were assayed for the presence ofperoxidase as described above in Part A, and the concentration of PAPwas calculated from the standard curve data.

The resulting elution profiles are presented in FIG. 10. The samplecontaining CBP2 and PAP exhibited a dramatic loss of PAP binding tosolid phase CBP2 on the column, while the sample containing LHRH and PAPshowed PAP binding to the column in the range normally observed. Thesedata show that PAP binding was dependent on CBP2.

If inactive CBP2 aggregates caused the low efficiency of PAP elution,then the same aggregates would effectively reduce the CBP2 concentrationand the inhibition of PAP binding in the pre-incubation studies wouldrequire very high CBP2 concentrations. This was not the case, however,since similar concentrations were used for column elution as for thepre-incubation studies. Also, another attempt at PAP inhibition in thepre-incubation studies using 156 μg/ml CBP2 demonstrated inhibition ofPAP binding (approximately 40%). Thus, the effect of aggregates, if theyformed, was of little significance.

2. In order to address the possible effects of acetic acid exposure onPAP, a 20 μg/ml sample of PAP was dissolved in either 2% (v/v) aceticacid or C1q buffer and incubated for 3 hours. Aliquots from both sampleswere then assayed for peroxidase activity. No significant differences inthe two samples were observed. Therefore, an adverse effect of aceticacid on HRP did not account for the results.

3. Although the data indicated that PAP binding was specific anddependent on CBP2, the PAP binding could have been due to a nonspecificinteraction of the column with the horseradish peroxidase (HRP) of PAP.Thus, a solution of HRP in C1q buffer was loaded, eluted and assayed forperoxidase as described for the PAP experiments. The linear regressioncalculations of the resulting profile indicated no significant bindingof HRP (only about 7%) to the CBP2 column.

4. The extent of nonspecific binding of PAP to the HiPAC™ matrix wasdetermined. Control columns coupled with either LHRH or No peptide wereused in place of the CBP2, in order to assess the extent of nonspecificbinding. In the same manner as described for the CBP2 column, PAP wereloaded and eluted either from the LHRH or No peptide column. The columnfractions were assayed for the presence of immunoglobulin using theenzyme immunoassay described above in Part B. Linear regression analysisof the standard curves allowed the calculation of the amount of materialin the flow-through and acid-eluted fractions.

The results are shown in Table 1 above. The LHRH column bound 4.8% ofthe total PAP loaded on the column, while the No-peptide column bound 8%of the total PAP. Therefore, the nonspecific binding of PAP to the CBP2column can account for, at most, a small fraction of the total PAPbinding observed.

E. Conclusion

The data show that the binding of PAP to the CBP2 column was specificfor immune complexes and dependent on CBP2, and that CBP2 wasinteracting with the immunoglobulin components of the PAP complex.

Example 5 Aggregated Human IgG Binding To The CBP2 Column

Human IgG was aggregated with alkali according to the method of Jones etal., J. Immunol. Meth., 53, 201-208 (1982). Alkali-aggregated IgGpossesses C1q binding activities similar to those of native immunecomplexes, thereby satisfying the essential criterion for immune complexmodels.

A 1 ml sample of 100 μg/ml of the aggregated IgG diluted in C1q bufferwas loaded onto a CBP2 column (prepared as described in Example 3) thathad been equilibrated with 3 ml C1q buffer. The sample was circulatedthrough the column for 15 min and then eluted with 21 ml C1q buffer,followed by 3 ml 2% (v/v) acetic acid, and then by 6 ml C1q buffer.

The fractions were assayed for immunoglobulin by placing duplicate 50 μlaliquots from each fraction and 50 μl of coating buffer into the wellsof a microtiter plate and incubating the plate 2 hours at 37° C. Theuncoated surfaces were blocked by adding 340 μl/well of PBSC followed byincubation of the plate for 1 hour at 37° C. Next, the wells were washed3 times with PBSCT. Goat anti-human IgG (Heavy and Light chain specific)F(ab')₂ fragments labeled with horseradish peroxidase (OrganonTeknika/Cappel, West Chester, Pa.) was added to the wells (100 μl/well)at a 1:30,000 dilution in PBSCT, and the plate was incubated for 2 hoursat 25° C. The wells were washed 3 times with PBSCT, OPD substrate bufferadded (100 μl/well), and the resultant colored product was measured at414 nm with a Titertek Multiskan plate reader. A standard curve using aportion of the sample loaded on the column was used in the assay inorder to quantitate the amount of IgG in each column fraction.

The elution profile for the aggregated IgG showed two major peaks (FIG.11). The aggregates not bound by the column were readily eluted with C1qbuffer and were found to make up the first, flow-through peak. Theflow-through material was calculated to comprise 5% of the totalaggregated IgG added to the column. The aggregates bound to the columnwere eluted with the acetic acid wash and constituted the larger secondpeak (FIG. 11). Linear regression calculations indicated that 95% of theaggregated IgG loaded on the column was bound to the column and elutedwith the acetic acid wash. Based on these data, the efficiency of theCBP2 column for binding aggregated IgG was considered quite high.

Next, a 1 ml sample of 400 μg/ml monomeric human IgG was diluted in C1qbuffer, loaded on a CBP2 column, eluted and assayed in the same fashionas described above for the aggregated human IgG. Also, a one ml samplecontaining a mixture of 100 μg/ml aggregated human IgG and 400 μg/mlmonomeric human IgG in C1q buffer was loaded, eluted, and assayed asdescribed above. The results are shown in FIG. 12 and Table 2 below.

                  TABLE 2    ______________________________________    Percent Of Added Material Bound to the Column    Sample   CBP2 Column LHRH Column                                    No Peptide Column    ______________________________________    Aggregated             96.7        18.8       26.3    IgG    Monomeric             31          N.D..sup.a N.D..sup.a    IgG    Aggregated +             95.9.sup.b  N.D..sup.a N.D..sup.a    Monomeric    IgG    ______________________________________     .sup.a Not Determined     .sup.b The value shown is the percentage of aggregated IgG bound. The     aggregates were biotinylated allowing for a separate determination (see     below).

The linear regression analysis calculations indicated that approximately31% of the total monomeric IgG loaded on the column was bound (see Table2). This amount of binding of the human monomeric IgG was quite highrelative to previous binding experiments using monomeric rabbit IgG.However, there was a significant difference in the amount of binding ofaggregated IgG and that of monomeric IgG.

The linear regression calculations for the mixture of aggregated andmonomeric human IgG determined that there were 100 μg of IgG in theacid-eluted fractions, indicating that the column bound almost all ofthe aggregated IgG. Although the calculated amount of IgG in theacid-eluted fractions closely corresponded to the amount of aggregatedIgG loaded onto the column, there was no evidence indicating whether ornot the aggregated IgG was actually the species binding to the column.It was, therefore, necessary to be able to distinguish the aggregatedIgG in a mixture of monomeric IgG and aggregates.

To do this, a 5 mg/ml preparation of aggregated human IgG was labeledwith sulfosuccinimididyl-6-(biotin-amido)hexanoate (NHS-LC-Biotin,Pierce Chemical Co., Rockford, Ill.) according to the method of Geudsonet al., J. Histochem. Cytochem., 27, 1131-39 (1979). The aggregated IgGhad 14% of the available primary amines biotinylated as determined bythe 2,4,6-trinitrobenzene sulfonic acid (TNBS) method described inFields, Meth. Enzymol., 25B, 464-68 (1972). Biotinylation of theaggregates using an N-hydroxysuccinamide ester adduct of biotin allowedthe rapid and specific labeling of the aggregates. Biotinylation hasbeen demonstrated to have minimal effects on the native conformationsand activities of biomolecules, and CBP2 +/- biotin showed no differencein its Protein A inhibitory activity. In addition, the avidin-biotininteraction is of a very high affinity (femptomolar in magnitude)providing a highly specific and extremely sensitive label for theaggregated IgG.

Next, a 1 ml sample containing biotinylated aggregated human IgG at 100μg/ml plus monomeric IgG at 400 μg/ml in C1q buffer was loaded, eluted,and the column fractions assayed for immunoglobulin as described above.The fractions were also assayed for biotinylated aggregates as follows.Duplicate 50 μl aliquots of each column fraction were mixed with 50 μlcoating buffer in the wells of a microtiter plate, and the plate wasincubated for 2 hours at 37° C. The uncoated surfaces of the wells wereblocked with PBSC. Avidin plus biotinylated horseradish peroxidase(Vector Laboratories, Inc., Burlingame, Calif.) (5 μl of each) dilutedin 1 ml PBSCT were incubated for 30 min at 25° C. and then diluted 1:15in PBSCT prior to adding 100 μl/well of the resulting complexes to theplate. The plate was incubated for 2 hours at 25° C., the wells washed 3times with PBSCT, and OPD substrate buffer added, and the coloredproduct measured at 414 nm in a Titertek Multiskan plate reader.

The results are shown in FIGS. 13A (assay for immunoglobulin) and 13B(assay for biotinylated aggregated IgG) and Table 2 above. Theconcentration of biotinylated aggregated IgG was calculated from thestandard curve and linear regression analysis, and it was found that95.6% of the biotinylated aggregated IgG loaded on the column was boundby the column (see Table 2), which indicates that immune complexes canbe bound to the CBP2 column specifically in the presence of monomericimmunoglobulin.

As a control, C1q was coated on the wells of a microtiter plate asdescribed in Example 2, and aliquots from each column fraction wereincubated with the solid phase C1q for 2 hours at 25° C. The wells werewashed 3 times with C1q buffer. Bound immune complexes were assayed asdescribed above using goat anti-human IgG F(ab')₂ fragments. Suchantibody fragments cannot be bound by C1q, since the fragments have noFc region.

The results of this assay of C1q binding activity showed that all of theC1q binding material was in the acid-eluted fractions, which are thefractions containing aggregated IgG that bound to the CBP2 column (seeFIG. 13C). The combined data show that the CBP2 column can bind humanimmune complexes specifically, and that the CBP2 column binds immunecomplexes which are bound by C1q.

The binding of aggregated IgG by the control columns is also shown inTable 2. Approximately 19% of the total aggregates added to the LHRHcolumn were bound to it, while approximately 26% of the total aggregatesbound to the No-peptide column. Although the nonspecific binding of theaggregate to the matrix is 19-26%, the binding of aggregated IgG to theCBP2 column (95% of total aggregated IgG) clearly cannot be explained bynonspecific effects alone. Based on the relatively high percentage ofnonspecific binding to the matrix, the monomeric IgG binding to thecolumn may be attributable to nonspecific binding.

In conclusion, the above data show that the CBP2 column binds immunecomplexes that are also bound by C1q and that the CBP2 column is bindingimmune complexes in a specific fashion.

Example 6 Binding Of Aggregated IgG and Serum Components To The CBP2Column.

Biotinylated aggregated IgG (100 μg/ml), prepared as described inExample 5, was diluted in normal human plasma which had been diluted1:20 with C1q buffer. A 1 ml sample of the aggregated IgG in dilutedhuman plasma was applied to a CBP2 column (prepared as described inExample 3) and eluted with C1q buffer, followed by a 2% (v/v) aceticacid wash to elute the bound material. The column fractions were assayedfor immunoglobulin using goat anti-human IgG F(ab')₂ as described inExample 5, and the results are shown in FIG. 14A. The biotinylatedaggregated IgG was detected with avidin-biotinylated-HRP complexes asdescribed in Example 5, and the results are shown in FIG. 14B. Finally,C1q binding activity was assessed as described in Example 5, and theresults are shown in FIG. 14C.

Nearly all of the biotinylated aggregates bound to the column as can beseen in FIGS. 14B and C. The calculated total aggregates bound was 98.2%of the amount added as determined from the linear regression analysis ofthe accompanying standard curve. Thus, the efficiency of immune complexbinding in dilute plasma was quite high.

In view of the success of this experiment, human serum samples weretested on the CBP2 column. The levels of immunoglobulin and immunecomplexes in the sera were determined prior to their passage over theCBP2 column to establish baseline readings. To do so, either Protein Aor C1q was coated on the wells of a microtiter plate as described inExample 2, and then 1:100 diluted serum samples were incubated with thesolid phase Protein A or C1q. Any bound material was detected using goatanti-human IgG F(ab')₂ fragments labeled with HRP as described inExample 5. A standard curve of aggregated IgG was also included witheach assay and provided a way to normalize readings between the twoassays (i.e., in aggregated IgG equivalents).

The calculated aggregated IgG equivalents for both the Protein A and C1qassays of the serum samples before passage over the column are presentedin Table 3. Ratios of the amounts of material bound by C1q to theamounts of material bound by Protein A, and expressed as percentages,are presented in FIG. 15. The Protein A assay showed that the sera hadconcentrations of immunoglobulin (IgG) ranging from 726.9 to 1336.2μg/ml, while the corresponding C1q assay showed immune complex (IC)levels ranged from 7.1 to 32.1 μg/ml (see Table 3).

                  TABLE 3    ______________________________________    Amounts of IgG or Immune Complexes (IC)    determined by assay (μg/ml)                     Protein A                              Clq    Serum Number     (IgG)    (IC)    ______________________________________    325              725.9    32.1    332              749.1    17.6    318              1183.1   17.3    335              1336.2   7.1    321              1175.7   11.0    ______________________________________

Next, the serum samples were diluted 1:20 with C1q buffer and loadedonto a CBP2 column. The column was washed with C1q buffer, and the boundmaterial eluted from the column with 2% (v/v) acetic acid. The columnfractions were assayed for immunoglobulin as described in Example 5.From the resulting elution profiles and the linear regression analysisof the standard curves, the amount of immunoglobulin passed over thecolumn (total IgG) and the amount of material bound (acid-elutedfractions) were determined, and the results are presented in Table 4.

                  TABLE 4    ______________________________________    Amounts of IgG (μg)            Total IgG    Acid-Eluted Fractions    Serum Number              Calculated.sup.1                         Actual.sup.2                                 Calculated.sup.1                                          Actual.sup.2    ______________________________________    325       36.3       126.8   1.61     4.73    332       37.5       49.4    0.88     2.87    318       59.2       33.3    0.87     1.72    335       66.8       84.77   0.36     5.08    321       58.8       40.5    0.55     4.59    ______________________________________     .sup.1 Calculated amount in serum before it was passed over the column     using Protein A and Clq binding assays.     .sup.2 Calculated in the present experiment using the assay for IgG.

To the acid-eluted fractions, 100 μl of 3.4M NaOH was added toneutralize the acetic acid. The column fractions were then divided intotwo groups (the flow-through and the acid-eluted fractions), pooled andthen dialyzed against PBS overnight at 4° C. The dialyzed fractions werethen aliquoted and stored at -70° C. until the samples could be analyzedas described below.

First, a 1 μg/ml solution of Protein A in coating buffer was incubatedin the wells of a microtiter plate (100 μl/well) for 2 hours at 25° C.The uncoated surfaces were blocked with PBSC as described in Example 2.Aliquots (100 μl/well) of both the acideluted and flow-through fractionswere incubated with the solid phase adsorbed Protein A for 2 hours at25° C. The wells were then washed 3 times with PBSCT, and goatanti-human IgG F(ab')₂ labeled with horseradish peroxidase diluted1:30,000 in PBSCT was added (100 μl/well). The plate was incubated for 2hours at 25° C. The wells were washed 3 times with PBSCT, followed bythe addition of OPD substrate buffer. The colored product was measuredat 414 nm using a Titertek Multiskan plate reader.

Also, C1q was coated onto the wells of a microtiter plate. A 10 μg/mlsolution of C1q in coating buffer was added to the wells of a microtiterplate (100 μl/well), and the plate was incubated for 2 hours at 25° C.The wells were blocked with PBSC as described in Example 2. Samples (100μl/well) from both the acid-eluted fraction and the flow-throughfraction were incubated with the solid phase C1q for 2 hours at 25° C.The wells were washed 3 times with PBSCT, and goat anti-human IgGF(ab')₂ was added at a 1:30,000 dilution in PBSCT (100 μl/well), and theplate was incubated for 2 hours at 25° C. PBSCT was used to wash thewells 3 times, OPD substrate buffer added, and the colored productmeasured at 414 nm in a Titertek Multiskan plate reader.

A standard curve of aggregated human IgG was used for both the C1q andprotein A binding assays. The use of a common standard curve facilitateda comparison of the results from the two assays, and results wereexpressed in aggregated IgG equivalents.

The amounts of immunoglobulin (Protein A binding material) and immunecomplexes (C1q binding material), expressed in aggregated IgGequivalents are presented in Table 5. Also, the amount of immunecomplexes as a percentage of total immunoglobulin is presented in FIG.16.

                  TABLE 5    ______________________________________    Amounts of IgG or Immune Complexes (IC) in    Column Fractions (μg/ml)              Flow-Through     Acid-Eluted                Protein A                         Clq       Protein A                                          Clq    Serum Number                (IgG)    (IC)      (IgG)  (IC)    ______________________________________    325         39.6     0.10      0.30   0.074    332         25.6     0.082     0.16   0.095    318         123.1    0.11      0.16   0.097    335         19.7     0.27      0.14   0.13    321         15.7     0.23      0.161  0.159    ______________________________________

The C1q binding activity detected in the flow-through fractions was asmall percentage of the total IgG as determined by Protein A binding(Table 5). The acid-eluted fractions had a much higher percentage of C1qbinding material when compared to the Protein A reactive material (Table5). In some cases it was as high as 98%. By comparing the resultspresented in FIG. 16 with the results in FIG. 15, it can be seen thatthe acid-eluted fractions were enriched with immune complexes (asdetected by the C1q assay).

The efficiency of the column the ratio of the total amount of C1q boundmaterial in the pooled acid-eluted fractions versus the amount of C1qbinding material in a 1 ml sample of 1:20 diluted serum before passageover the column! ranged widely from 23% to 109%. This variability may bedue to the fact that sera vary in their immune complex content due tothe variation from person to person. The CBP2 column and the C1q assaymay also be detecting different species of immune complexes. C1q hasvery distinct binding affinities for IgG subclasses and for IgM, but theimmunoglobulin specificity of the CBP2 column remains to be determined.

Example 7 HiPACT™ Fast Protein Liquid Chromatography (FPLC) Column

A. CBP2 Coupling to HiPAC™ FPLC Column.

CBP2 was coupled to an HiPAC™ FPLC column (dimensions of 0.6 mm×10 cm)in the following fashion. The column was equilibrated with 10 columnvolumes (18 ml) of 0.1M sodium citrate, pH 5.5, at a flow rate of 1ml/min at 25° C. A ligand solution of 1.4 mg/ml CBP2 diluted in 0.1Msodium citrate, pH 5.5, plus 20 mg/ml sodium cyanoborohydride wasprepared, and a 3 ml sample applied to the column. The sample wascirculated through the column for 20 min at 25° C. at a flow rate of 1ml/min using a peristaltic pump (Rainin Inc., Woburn, Mass.). The columnwas then washed with 20 ml of sodium citrate, pH 5.5, at 1 ml/minfollowed by 10 ml of 2M guanidine-HCl, and then 10 ml of 0.05M Tris, pH9.0, at the same flow rate. The column was then washed with 20 ml C1qbuffer and stored in C1q buffer for future use. This column had a bedvolume of 1.8 ml as compared to 0.8 ml for the LTQ column.

The extent of CBP2 coupling to the column was determined by directlyassaying the column with Ellman's reagent as described in Example 3. Theamount of CBP2 coupled to the column as determined by this assay was 756μg. The density of CBP2 in relation to the column volume was 420 μgCBP2/ml matrix. This is 20% less than the density obtained for the LTQcolumn, which was 525 μg CBP2/ml matrix.

B. Aggregated Human IgG Binding to the FPLC-CBP2 Column.

The CBP2/FPLC column was equilibrated with C1q buffer at a flow rate of1 ml/min for 15 min using a Beckmam HPLC (Model 421 controller and 112solvent delivery module, Palo Alto, Calif.). A 2 ml sample of 100 μg/mlaggregated human IgG (prepared as described in Example 5) diluted in C1qbuffer was loaded onto the column with C1q buffer at a flow rate of 1ml/min for 2 min. The flow rate was then increased to 2 ml/min, andmaintained for 20 minutes, at which time the flow rate was reduced to 1ml/min. The column was next washed with 2% (v/v) acetic acid at a flowrate of 1 ml/min for 12 min, followed by C1q buffer at a flow rate of 1ml/min for 6 min. The column eluate was collected at 1 minute intervalsand assayed for the presence of immunoglobulin as described in Example5.

The resulting profile is presented in FIG. 17. From the linearregression calculations, the concentration of aggregated IgG in eachcolumn fraction was determined. According to the calculations, 96% ofthe total aggregated IgG added to the column was found in theacid-eluted fractions. Therefore the column was able to bind immunecomplexes with a high efficiency.

In order to determine the specificity of the FPLC-CBP2 column for immunecomplexes, monomeric human IgG was loaded and eluted from the column andassayed as described above for the aggregated IgG. The linear regressioncalculations determined that 13% of the monomeric IgG loaded on thecolumn was bound to the column, indicating that the FPLC-CBP2 column wasin fact specific for immune complexes and more specific than the LTQcolumn which exhibited 31% binding of monomeric human IgG.

Next, biotinylated aggregated IgG at 100 μg/ml was added to human plasmadiluted 1:20 in C1q buffer, and a 2 ml aliquot of the sample was loadedand eluted from the CBP2 -FPLC column as described above and thenassayed for total IgG and biotinylated aggregated IgG as described inExample 6.

The resulting profiles are presented in FIG. 18. FIG. 18 shows extensivetrailing of the flow-through material, with a drop in IgG concentrationprior to the acid-elution fractions. The acid-elution fractions have apeak of IgG. The aggregated IgG profile in FIG. 18 shows that theaggregates remained bound to the column despite extensive flow-throughof excess IgG, until they were eluted by the acid wash. Based on linearregression analysis of the aggregated IgG profile, nearly all of thebiotinylated aggregated IgG was bound by the column. The data alsoindicated that the FPLC-CBP2 column could bind immune complexespreferentially in the presence of free monomeric IgG, in a fashionsimilar to the LTQ-CBP2 column.

C. Human Serum Components Binding to the FPLC-CBP2 Column.

Sera were diluted 1:20 in C1q buffer and loaded and eluted as describedabove, and the column fractions assayed as described in Example 6. Theresults are presented in Tables 7 and 8 and FIG. 19.

Table 7 shows the total IgG in the initial serum sample ("calculated"),the total IgG found in the eluted fractions after the serum samples werepassed over the column ("actual"), the amount of immune complexes boundto the column and eluted with the acid wash (acid-eluted fractions)("actual"), and the amount of immune complexes in the initial sample("calculated"). FIG. 19 shows the ratio of C1q binding material toProtein A binding material for each serum sample based on the values inTable 8 which shows the amounts of Protein A and C1q binding material inthe pooled flow-through and acid-eluted fractions.

                  TABLE 7    ______________________________________                         Acid-Eluted            Total IgG (μg)                         Fractions (μg)    Serum Number              Calculated.sup.1                         Actual.sup.2                                 Calculated.sup.1                                          Actual.sup.2    ______________________________________    325       72.6       254.5   1.76     0.376    332       75.0       130.8   1.76     3.86    318       118.2      138.62  1.74     2.33    335       133.6      141.64  0.72     1.71    321       117.6      280.79  1.10     3.02    ______________________________________     .sup.1 Calculated amount in serum before passage over the column as     determined by binding to Protein A or Clq.     .sup.2 Calculated amount in fractions after passage over the column;     calculated using assay for IgG.

                  TABLE 8    ______________________________________    Amounts of IgG or IC in Column Fractions (μg/ml)             Flow-Through     Acid-Eluted               Protein A                        Clq       Protein A                                         Clq               Binding  Binding   Binding                                         Binding    Serum Number               (IgG)    (IC)      (IgG)  (IC)    ______________________________________    325        83.2     0.0795    0.188  0.0458    332        42.7     0.342     0.68   0.0291    318        73.2     0.0867    0.497  0.224    335        27.0     2.45      0.270  0.116    321        82.9     1.45      0.584  0.178    ______________________________________

Based on the data, the FPLC-CBP2 column appeared to bind immunecomplexes in a similar fashion as the LTQ-CBP2 column. The FPLC-CBP2column, like the LTQ column, also demonstrated a significant enrichmentfor C1q binding material in the acid-eluted fractions.

The FPLC-CBP2 column efficiencies differed significantly from the LTQcolumn efficiencies with regards to sera 325 and 332 (Table 9). Bothcolumns demonstrated high binding efficiencies for sera 335 and 321,while serum 318 was bound with a moderate efficiency by both columns.

                  TABLE 9    ______________________________________                 Efficiency                   CBP2-LTQ  CBP2-FPLC    Serum Number   Column    Column    ______________________________________    325            23%       8.54%    332            54%       9.94%    318            56%       78.1%    335            109%      96.7%    321            87%       97.2%    ______________________________________     .sup.a The efficiency of binding was calculated as the percentage of     actual ICs in the acideluted fractions over the calculated ICs in the     sample added to the column.

In conclusion, based on similar trends in immune complex bindingefficiencies and enrichments, the FPLC-CBP2 column was determined to bea reasonable scaled-up version of the LTQ-CBP2 column.

Example 8 Inhibition Of Complement-Mediated Lysis Of Cells

A. Synthesis Of Peptides

Several additional peptides were prepared as described in Example 1. Thefirst of these peptides, called CBP3 herein, has the following sequence:

    Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile    1               5                   10    Leu His Cys            15                      SEQ ID NO:4!

Residues 1-14 of SEQ ID NO:4 are from the sequence of that portion ofProtein A that binds to immunoglobulin.

The second peptide, referred to herein as AEK, is a portion of the CH2region of immunoglobulin to which C1q binds. This peptide has thesequence:

    Ac-Ala Glu Ala Lys Ala Lys Ala-NH.sub.2  SEQ ID NO:5!       1               5

The third peptide has the sequence:

    Ser Arg Phe Leu Thr Ser  SEQ ID NO:6!    1               5

This peptide was used as a hydrophilic control peptide in the lysisassays described below.

A fourth peptide, referred to herein as CBP1, has the followingsequence:

    Lys Leu Lys Glu Leu His Glu Phe Ile Lys Glu Leu     1               5                  10    Leu Leu Cys            15                      SEQ ID NO:7!

This peptide was also used as a control peptide in the lysis assaysbecause of its similar size, but different sequence from, CBP2 and CBP3.

B. Sheen Red Blood Cell Lysis Assay (SRBC Assay)

To perform this assay, sheep erythrocyes (SRBCs) are "sensitized" by theaddition of anti-SRBC antibody. Addition of serum as a source ofcomplement results in C1 binding and activation of the complement systemwith resultant lysis of the erythrocytes. The released hemoglobin ismeasured spectrophotometrically and is related to the amount ofcomplement activation.

SRBCs were purchased from Bio Whittaker, Walkersville, Md. They werediluted to 0.28% cells/ml in Tris-buffer saline containing gelatin TBSG,50 mM Tris-HCl (Mallinckrodt Specialty Chemicals Co., Paris, Ky.) and150 mM NaCl (Aldrich Chemical Co., Milwaukee, Wis.) plus 2% swine skingelatin (Type 1, Sigma Chemical Co., St. Louis, Mo.)!.

Hemolysin reagent (anti-sheep erythrocyte stromata serum) was purchasedfrom Bio Whittaker, Walkersville, Md. It was diluted 1:400 in TBSG.

Human serum was used as the source of complement. Blood was drawn from avolunteer into 6 ml Vacutainer brand serum separation tubes (BectonDickinson, Franklin Lakes, N.J.), and the serum was separated bycentrifugation at 1500×g for 10 min at room temperature. Serum wasstored in small aliquots at -70° C. The serum was titered for complementactivity by using serial dilutions in TBSG added to SRBCs and hemolysin.A serum dilution was used in subsequent assays that gave between 40-80%of total SRBC lysis.

The diluted SRBCs (50 μl) were added to wells of a Corning 96-well, flatbottomed, polystyrene, non-coated plate (Corning Glass Works, Corning,N.Y.). Then, 50 μl of diluted hemolysin was added, followed by 50μl ofvarious inhibitors. The well contents were gently mixed using amultichannel pipette, and the plate was incubated for 30 min at 37° C.The diluted serum (50 μl) was added to the mixture of SRBCs, hemolysinand inhibitors, and each well was gently mixed. The plate was incubatedfor 60 min at 37° C. The plate was then centrifuged at 500×g at roomtemperature for 5 min, after which 100 μl of supernatant were removedfrom each well and transferred to another 96-well Corning plate. Therelative amounts of hemoglobin were quantitated at 405 nm using aCoulter Microplate Reader (Hialeah, Fla.). Total cell lysis wasdetermined by pipetting an aliquot of the stock SRBCs into 200 μl of H₂O, removing 100 μl of supernatant and determining the OD at 405 nm.

Total complement activity of the diluted human serum was calculated asthe OD at 405 nm of human serum without any additions minus OD at 405 nmof an incubation mixture without human serum or with heat-inactivatedhuman serum (treated for 1 hour at 56° C.). The percent inhibitionobtained with various inhibitor substances was calculated as the totalcomplement activity (100%) minus the percent complement activity in thepresence of inhibitor. All assay points were determined in duplicate ortriplicate and analyzed using Microsoft Excel. The results are shown inTables 10 and 11 below.

C. Chromium Release Assay

Culture plates (Falcon 24-well plates, Becton Dickinson, Lincoln Park,N.J.) were coated by addition of 0.5 ml/well of 2% gelatin (Type B, frombovine skin, Sigma Chemical Co.) diluted 1:10 in Hanks buffered saltsolution (Gibco BRL, Grand Island, N.Y.), followed by incubation for 15min at room temperature. The gelatin solution was removed, and 125×10³pig aorta endothelial cells (PAECs, Cell Systems, Kirkland, Wash.) in 2ml of cell culture medium (CCM, Dulbecco's modified Eagle's medium(DMEM) with high glucose (Gibco BRL) containing 20% defined fetal bovineserum (FBS, HyClone Laboratories, Logan, Utah) that had been heatinactived for 30 min at 56° C., 1%(v/v) L-glutamine (200 mM, Gibco BRL)and penicillin G sodium (10,000 units/ml)/streptomycin sulfate (10,000μg/ml in 0.85% saline, Gibco BRL)! were added per well. The plate wascentrifuged in a model GS-6R Beckman table top centrifuge at 20×g for 2min with the brake off to settle the cells onto the gelatin in a evenmonolayer, after which the plate was incubated at 37° C. in 5% CO₂ -95%air. After 4 hours, cell attachment had occurred, and the CCM wasremoved and replaced with 2 ml of fresh CCM. In 2-4 days the PAECs hadgrown to 80-100% confluence and were used in the complement mediatedchromium release assay.

The PAECs were washed 3 times with 1 ml of serum free medium (SFM),leaving the SFM on the cells for 5 min each time. Chromium radioisotope(⁵¹ Cr, NaCrO₄, Amersham Corp., Arlington Heights, Ill.) was diluted inSFM to give 40 μCi/ml, and 100 μl were added to each well of the PAECculture plate. The plate was incubated for 2 hours at 37° C. in 5% CO₂-95% air. The wells were washed three times with 1 ml of SFM. Then, 1 mlof PBS was added to each well, and the plate was incubated at 37° C. for30 min to help reduce ⁵¹ Cr background.

The various peptide inhibitors (diluted in SFM) were added (300 μl/well)and incubated with the PAECs at 37° C. for 30 min. Undiluted human serum(HS, 100 μl) or 100 μl of heat inactivated human serum (HIHS) was addedto each well, and the plate was incubated at 37° C. for 1 hour.Supernatant (200 μl) was removed from each well and added to 200 μl ofscintillation cocktail (OptiPhase `Supermix`, Wallac Inc., Turku,Finland) previously added to each well of a 24-well polyethyleneterephtalate reading plate (Wallac Inc.), and the plate was sealed withan acetate plate sealer (Dynatech Laboratories, Chantilly, Va.). Thereading plate was placed on a shaker for 5 min to obtain a homogeneousmixture. Radioactivity (CPMs) of the wells was counted for 2 min usingthe 1450 MicroBeta PLUS plate reader (Wallac Inc.).

Total counts of ⁵¹ Cr were determined from 200 μl aliquots taken fromthe wells to which 100 μl of 5% Triton X-100 (Sigma Chemical Co.) hadbeen added. Total complement mediated lysis was determined from wellstreated with HS alone, and background counts were determined from wellstreated with HI-HS alone or with peptide alone (no HS). The averagebackground was subtracted from all well counts, and the percentinhibition calculated as total lysis minus lysis with inhibitor/totallysis multiplied by 100. All experimental points were obtained intriplicate, and the data were analyzed in the same manner as the datafrom the SRBC assays. The results are presented in Tables 10 and 11below.

C. Immunohistochemical Staining Of PAECs For Binding Of Human C1q

PAECs (1×10⁴) in 1 ml of CCM were added to each well of a 4-chamberculture slide (Labtek, Nunc, Naperville, Ill.), and the cells were grownto confluence (incubation for 3 days at 37° C. in 5% CO₂ -95% air). Themedium was removed, and the slide was washed with PBS (10 mM phosphatebuffered saline, pH 7.4, containing 120 mM NaCl and 2.7 mM KCl, SigmaChemical Co.) in a swirling motion for 5 sec. This was repeated withdistilled water and 100% ethanol. The slide was tapped dry and immersedin pre-chilled (-20° C.) acetone for 20 min and then air dried.

HI-HS was added to the wells, and the slide was incubated for 15 min at37° C. to allow human IgG and IgM antibodies to bind to PAEC epitopes.The HI-HS was removed, and the wells were washed 3 times with PBS. C1qor C1q plus peptide in PBS containing 1% bovine serum albumin (BSA,Sigma Chemical Co.) (PBSA) was then added, and the slide was incubatedfor 15 min at room temperature. The solutions were removed, and thewells were washed 3 times with PBS. Blocking serum was added 10% donkeyserum (The Binding Site, Birmingham, England) in PBS!, and the slide wasincubated for 20 minutes at room temperature. The donkey serum wasremoved, the wells were washed with PBS, sheep anti-human C1q (BiodesignInt., Kennebunk, Me.) diluted 1:200 in PBSA was added, and the slide wasincubated for 1 hour at room temperature. After washing the wells withPBS, fluorescein isothiocyanate (FITC) conjugated donkey anti-sheep IgG(The Binding Site, Birmingham, England) diluted 1:200 in PBS was added,and the slide was incubated for 30 min at room temperature. The wellswere again washed with PBS. The slide was then counterstained using0.01% methyl green (Sigma Chemical Co.). Then, the wells were washedwith PBS, and 1% eriochrome black (Sigma Chemical Co.) was added for 10sec. The wells were washed with PBS, and the slide coverslipped withAquamount (Scientific Products, McGaw Park, Ill.). Staining of the cellswas analyzed on a Zeiss Axioskop (Carl Zeiss, Oberkochen, Germany) witha wide band FITC filter.

E. Results

The peptides CBP2 and CBP3 were effective inhibitors of complementactivation as measured in the SRBC and PAEC lysis assays. CBP2 showednearly complete inhibition of lysis of sensitized SRBC using human serumas complement source at 500 μM concentration and 13.5% inhibition at62.5 μM (see Table 10). The data are the results of five differentexperiments at various times. The inhibition values and standarddeviations at the various points listed in Table 10 were evaluated forstatistical significance of the differences. As listed in the table, pvalues of <0.05 were obtained for the composite values of the fiveexperiments comparing inhibitions obtained with adjacent concentrationsof CBP2, increasing the probability of the validity of the differencesobserved.

CBP2 was also effective in a dose dependent manner in inhibitingcomplement-mediated lysis of PAECs by human serum containingxenoreactive natural antibodies and complement (Table 10). A maximuminhibition of 87.2% was achieved at the highest concentration of CBP2used (500 μM) and 10.6% inhibition at 62.5 μM.

CBP3 also showed complement inhibiting activity in the SRBC lysis assay(Table 11). The data are the averages of the results of five differentexperiments. At the highest concentration tested (1 mM), 83.6%inhibition was obtained. The CBP3 preparation seemed to be about half aseffective on a molar basis as CBP2.

Several other peptides exhibited no inhibitory activity in either theSRBC or the PAEC assays at the highest concentration (1 mM) tested.These peptides included SEQ ID NOS: 5, 6 and 7 (see above).

                  TABLE 10    ______________________________________    Inhibition of Complement-Mediated    Lysis With Various Concentrations of CBP2                  % Inhibition*                             % Inhibition*                  SRBC Assay PAEC Assay     CBP2! in μM                  +/- S.D.   +/- S.D.    ______________________________________    31            0.0 +/- 0.0                             2.0 +/- 3.1    62.5          13.5 +/- 9.3                             10.6 +/- 13.5    125           45.2 +/- 30.8                             25.0 +/- 14.8    250           78.0 +/- 18.3                             64.4 +/- 8.4    500           95.8 +/- 1.9                             87.2 +/- 3.8    1000          97.4 +/- 2.7                             Not Determined    ______________________________________     *ANOVA analysis of the differences of inhibition with the different     concentrations of CBP2 yielded a p value of <0.0001

                  TABLE 11    ______________________________________    Inhibition of Complement-Mediated SRBC    Lysis With Various Concentrations of CBP3     CBP3! in μM                  % Inhibition +/- S.D.*    ______________________________________    1000          81.8 +/- 3.0    800           64.3 +/- 1.5    675           57.7 +/- 3.5    500            38.2 +/- 25.9    325           33.3 +/- 4.2    250            21.6 +/- 21.1    125           0    ______________________________________     *ANOVA analysis of the differences of inhibition with the different     concentrations of CBP3 yielded a p value of <0.0001.

The immunohistochemical staining showed that CBP2 at concentrations of4.7 and 2.4 nM completely inhibited C1q binding to PEACs incubated withserum containing xenoreactive natural antibodies, and only trace bindingof C1q was seen at a CBP2 concentration of 1.2 mM. PAECs reacted withC1q without CBP2 showed strong fluorescence for C1q binding. Theseresults indicate that the complement activation inhibitory activity ofCBP2 was at the C1q-Ig binding step.

Example 9 Heterotopic Heart Transplantation

Four to six week old guinea pigs (GP, Harlan, Indianapolis, Ind.) heartswere transplanted into C6 deficient PVG rats (B&K, Kent, Wash.) by amodification of the technique described by Ono and Lindsay (Journal ofThoracic Cardiovascular Surgery, 57, 225, 1969). Seven of the ratsreceiving GP hearts were not treated, 8 additional rats receiving GPhearts were treated with a single pre-transplant intravenous injectionof a 3 ml cocktail of CBP3 SEQ ID NO:4! (0.67 mg/ml) and 1.03 mg/ml ofAEK SEQ ID NO:5!. CBP3, 15.4 mg, was dissolved in 1.1 ml of 60% DMF inH₂ O and AEK, 10 mg, was dissolved on 0.5 ml H₂ O. Two hundred μl ofCBP3 and 100 μl of AEK were added to 2.7 ml of 1:5 diluted (in PBS)heat-inactivated PVG C6 deficient rat serum. The GP transplanted heartswere monitored several times daily, and the times noted when the heartsstopped beating.

Two additional PVG C6 deficient rats were transplanted with GP hearts.The rats were fitted subdermally with a model 2ML4 ALZET osmotic pump(ALZA Corp., Palo Alto, Calif., mean pumping rate of 2.51 μl/hour)filled with 2.3 ml solution of CBP2 (15.6 mg of CBP2 dissolved in 1.5 mlof 50 mM Na₂ HPO₄ plus 0.15M NaCl, pH 9.0, and 0.8 ml heat-inactivatedPVG C6 deficient rat serum) on day 1. On day 2 the rats were injectedintravenously with 2.8 ml of a solution containing 15.6 mg CBP2dissolved in 1.8 ml of the Na₂ HPO₄ -NaCl buffer plus 0.9 ml ofheat-inactivated PVG rat serum.

Both CBP2 and the combination of CBP3 and AEK prolonged the survival ofthe GP hearts transplanted into the PVG C6 deficient rats (see Table 12below). For the 7 rats that did not receive any treatment, the averagegraft survival was 1.2+/-0.2 days. For the 8 rats that received GPhearts and were treated with CBP3 plus AEK, the average graft survivalwas 2.9+/-0.6 days (p=0.016 versus control rats). For the 2 rats treatedwith CBP2, the grafts survived for 4 and 5 days, respectively.

In a separate experiment, two C6 deficient PVG rats were transplantedwith GP hearts. Each rat had a 2ML1 ALZET osmotic pump (mean pumpingrate of 10 μl/hour) implanted the day before the transplants. In onerat, the pump was filled with 30 mg CBP2 dissolved in a 2.3 ml mixtureof 1.5 ml 0.1M Na₂ HPO₄ -0.15M NaCl plus 0.8 ml heat inactivated C6deficient PVG rat serum. This rat was also given an intravenousinjection of 15.7 mg of CBP2 dissolved in 1.33 ml of a mixture of 1.0 ml0.1M Na₂ HPO₄ -0.15M NaCl plus 0.33 ml heat inactivated C6 deficient PVGrat serum. The second rat was treated in the same manner as the firstrat, except that the pump contained 2.3 ml of the buffer-serum mixturewithout added CBP2, and the rat was given an intravenous injection of1.33 ml of the buffer-serum mixture without added CBP2.

The GP heart in the rat treated with buffer plus serum remainedfunctional for 3 days. The GP heart in the rat that received CBP2remained functional through the eighth day after transplant. See Table12 below.

                  TABLE 12    ______________________________________                              Graft                              Survival    Number Of Rats Treatment  (Days)    ______________________________________    7              Buffer + serum                              1.2 +/- 0.2    8              CBP3 + AEK 2.9 +/- 0.6    2              CBP2       4, 5                   (pump 2ML4)    1              Buffer + Serum                              3    1              CBP2       8                   (pump 2ML1)    ______________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  8    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  12 amin - #o acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  unknown    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 1:    - Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Se - #r Leu    #                 10    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  12 amin - #o acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  unknown    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 2:    - Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Al - #a Thr    #                 10    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  15 amin - #o acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  unknown    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 3:    - Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Al - #a Thr Leu Leu    #                 10    - Cys    15    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  15 amin - #o acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  unknown    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 4:    - Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Gl - #u Ile    #                 10    Leu His Cys            15    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  7 amino - # acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  unknown    -     (ix) FEATURE:    #Xaa is   (D) OTHER INFORMATION:                   acetyl al - #anine    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 5:    - Xaa Glu Ala Lys Ala Lys Ala                     5    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  6 amino - # acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  unknown    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 6:    - Ser Arg Phe Leu Thr Ser                     5    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  15 amin - #o acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  unknown    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 7:    - Lys Leu Lys Glu Leu His Glu Phe Ile Lys Gl - #u Leu Leu Leu    #                 10    Cys    15    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  14 amin - #o acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:              (D) TOPOLOGY:  unknown    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 8:    - Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Gl - #u Ile    #                 10    Leu His    __________________________________________________________________________

We claim:
 1. A method comprising:transplanting tissue into a mammal;providing a synthetic peptide or C1q fragment that inhibits binding of aC1q molecule to an immunoglobulin molecule, the peptide or fragmentcomprising the sequence:

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr     1               5                  10                                        SEQ ID NO 2!; and

administering to the mammal an effective amount of the peptide orfragment so that the tissue survives in the mammal for a period of timelonger than it would in the absence of the administration of the peptideor fragment.
 2. The method of claim 1 wherein the transplanted tissue isa xenotransplant.
 3. The method of claim 1 wherein the synthetic peptideis administered to the mammal.
 4. The method of claim 3 wherein thepeptide has the sequence:

    Leu Glu Gln Gly Glu Asn Val Phe Leu Gln Ala Thr     1               5                  10    Leu Leu Cys.            15                      SEQ ID NO 3!


5. The method of claim 1 wherein the C1q fragment is administered to themammal.
 6. A method comprising:transplanting tissue into a mammal;providing a synthetic peptide or Protein A fragment that inhibitsbinding of a C1q molecule to an immunoglobulin molecule, the peptide orfragment comprising the sequence:

    Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile    1               5                   10    Leu His;                      SEQ ID NO:8!    and

administering to the mammal an effective amount of the peptide orfragment so that the tissue survives in the mammal for a period of timelonger than it would in the absence of the administration of the peptideor fragment.
 7. The method of claim 6 wherein the transplanted tissue isa xenotransplant.
 8. The method of claim 6 wherein the synthetic peptideis administered to the mammal.
 9. The method of claim 8 wherein thepeptide has the sequence:

    Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile    1               5                  10    Leu His Cys.            15                      SEQ ID NO:4!


10. The method of claim 6 wherein the Protein A fragment is administeredto the mammal.